U.S. patent number 11,382,931 [Application Number 16/315,153] was granted by the patent office on 2022-07-12 for methods and composition for producing and using immune cells and stem cells for cell-based therapies.
This patent grant is currently assigned to University of Southern California. The grantee listed for this patent is University of Southern California. Invention is credited to Preet M. Chaudhary.
United States Patent |
11,382,931 |
Chaudhary |
July 12, 2022 |
Methods and composition for producing and using immune cells and
stem cells for cell-based therapies
Abstract
Described herein are methods for selecting lymphocytes for
adoptive cell therapy based on P-glycoprotein expression and
compositions comprising same.
Inventors: |
Chaudhary; Preet M. (Toluca
Lake, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
University of Southern California |
Los Angeles |
CA |
US |
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Assignee: |
University of Southern
California (Los Angeles, CA)
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Family
ID: |
1000006425690 |
Appl.
No.: |
16/315,153 |
Filed: |
July 14, 2017 |
PCT
Filed: |
July 14, 2017 |
PCT No.: |
PCT/US2017/042248 |
371(c)(1),(2),(4) Date: |
January 03, 2019 |
PCT
Pub. No.: |
WO2018/013993 |
PCT
Pub. Date: |
January 18, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20190209614 A1 |
Jul 11, 2019 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62362497 |
Jul 14, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
35/00 (20180101); A61K 35/17 (20130101); C12N
5/0646 (20130101); C12N 5/0635 (20130101); C12N
5/0636 (20130101); A61P 37/06 (20180101); A61P
31/18 (20180101); C12N 5/0647 (20130101); C12N
2503/02 (20130101); Y02A 50/30 (20180101); C12N
2529/10 (20130101); C12N 2523/00 (20130101) |
Current International
Class: |
A61K
35/17 (20150101); C12N 5/0789 (20100101); C12N
5/0783 (20100101); A61P 37/06 (20060101); C12N
5/0781 (20100101); A61P 31/18 (20060101); A61P
35/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Wittmann-Regis, Agnes, International Preliminary Report on
Patentability and Written Opinion, The International Bureau of
WIPO, PCT/US2017/042248, dated Jan. 24, 2019. cited by applicant
.
Guimond et al., "P-glycoprotein targeting: a unique strategy to
selectively eliminate immunoreactive T cells," Blood, vol. 100, No.
2, pp. 375-382, Jul. 2002. cited by applicant .
Guo et al., "Chemoprotection effect of retroviral vector encoding
multidrug resistance 1 gene to allow intensified chemotherapy in
vivo," Cancer Chemother. Pharmacol., vol. 58, No. 1, pp. 40-49,
Nov. 2005. cited by applicant .
Ward et al., "Retroviral transfer and expression of the human
multiple drug resistance (MDR) gene in peripheral blood progenitor
cells," Clin Cancer Res., vol. 2, No. 5, pp. 873-876, May 1996.
cited by applicant .
Copenheaver, Blaine R., International Search Report and Written
Opinion, U.S. Patent & Trademark Office, PCT/US2017/042248,
dated Oct. 2, 2017. cited by applicant .
Bayer, Martin, Search Report, Application No. 17828580.5, European
Patent Office, dated Feb. 20, 2020. cited by applicant .
Chaudhary P M et al., "Expression and activity of P-glycoprotein, a
multidrug efflux pump, in human hematopoietic stem cells", Cell,
vol. 66, No. 1, Jul. 12, 1991, pp. 85-94. cited by applicant .
D'Alessio, F. et al., "Polychromatic flow cytometry analysis of
CD34+ hematopoietic stem cells in cryopreserved early preterm human
cord blood samples", Cytometry Part A, vol. 79A, No. 1, Nov. 10,
2010, pp. 14-24. cited by applicant .
Mciver, Zachariah A. et al., "Selective photodepletion of
malignantT cells in extracorporeal photopheresis with
selenorhodamine photosensitizers", Bioroganic & Medicinal
Chemistry, vol. 24, No. 17, Jun. 2, 2016, pp. 3918-3931. cited by
applicant .
Perruccio et al., "Photodynamic purging of alloreactive T cells for
adoptive immunotherapy after haploidentical stem cell
transplantation", Blood Cells, Molecules and Diseases, vol. 40, No.
1 , Nov. 5, 2007, pp. 76-83. cited by applicant.
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Primary Examiner: Belyavskyi; Michail A
Attorney, Agent or Firm: Gavrilovich, Dodd & Lindsey
LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
The application is a U.S. National Stage Application filed under 35
U.S.C. .sctn. 371 and claims priority to International Application
No. PCT/US2017/042248, filed Jul. 14, 2017, which application
claims priority under 35 U.S.C. .sctn. 119 to U.S. Provisional
Application Ser. No. 62/362,497, filed Jul. 14, 2016, the
disclosures of which are incorporated herein by reference.
Claims
What is claimed is:
1. A method for isolating T cells and/or NK cells suitable for
adoptive cell therapy, comprising: (a) obtaining a sample; (b)
optionally enriching the sample for T cells and/or NK cells; and
(c) isolating p-glycoprotein positive (Pgp.sup.+) T cells and/or NK
cells from the sample, so as to obtain a fraction enriched in
Pgp-positive T cells and/or NK cells by contacting the sample with
(i) a cytotoxic drug that is a substrate of Pgp, (ii) exposing the
sample to at least one primary antibody or antibody-like moiety
specific to p-glycoprotein, and/or (iii) exposing them to serum
starvation, thereby isolating T cells and/or NK cells suitable for
adoptive cell transfer therapy.
2. The method of claim 1, wherein the at least one cytotoxic drug
is any one or more of vincristine, vinblastin, doxorubicin,
daunorubicin, taxol, paclitaxol, etoposide, mitoxantrone,
actinomycin-D, or combinations thereof.
3. The method of claim 1, wherein the at least one primary antibody
or antibody-like moiety is conjugated to at least one fluorescent
label or at least one magnetic label or biotin.
4. The method of claim 1, further comprising optionally staining
the sample with at least one secondary antibody.
5. The method of claim 4, wherein the at least one secondary
antibody is conjugated to at least one fluorescent label or at
least one magnetic label or biotin.
6. The method of claim 1, wherein the isolating of the Pgp-positive
cells from the sample is performed by any one or more methods
selected from immunofluorescent methods, immunomagnetic methods,
immunoaffinity methods, or combinations thereof.
7. The method of claim 1, wherein the fraction enriched in
Pgp-positive cells contains less than 50% Pgp-negative cells.
8. The method of claim 1, wherein the Pgp-positive cells are
further genetically modified so as to obtain genetically modified
Pgp-positive cells.
9. The method of claim 8, wherein the genetically modified
Pgp-positive cells are selected from the group consisting of T
cells and/or NK cells.
10. The method of claim 1, wherein Pgp-positive T cells are further
genetically modified to express at least one chimeric antigen
receptor, T cell receptor, synthetic immune receptor, chimeric T
cell receptor, or other genetic element so as to obtain genetically
modified Pgp-positive T cells.
Description
TECHNICAL FIELD
Provided herein are methods for selecting lymphocytes for adoptive
cell therapy based and methods of using such lymphocytes.
BACKGROUND
Immunotherapy using adoptive transfer of tumor-specific T cells and
chimeric antigen receptor (CAR) and T cell receptor (TCR) modified
T cells mediates durable and complete disease regression in some
patients with metastatic cancer. Recent studies have suggested that
metabolism supports and drives many basic features of T cells,
including cellular activation, proliferation, differentiation,
effector function, and antitumor immunity. This has led to a
growing interest in leveraging this understanding to improve the
efficacy of T cell transfer therapies, such as adoptive transfer
immunotherapy in the treatment of cancer.
Although there is increasing evidence that metabolism can affect
the survival and antitumor function of T cells, identifying a
simple and clinically feasible method to isolate T cells with
favorable metabolic features has proved challenging. Because
mitochondria are the central metabolic organelle in cells, Sukumar
et al. (Cell Metabolism, 23:63-76, 2016) hypothesized that the
measurement of a single mitochondrial-associated parameter may help
to identify T cells with a favorable bioenergetic profile that can
survive in vivo for long periods after adoptive transfer for T
cell-based immunotherapy, such as CAR-T, TCR and chimeric T cell
receptor (cTCR) based therapies.
Sukumar et al. described a clinically feasible method to isolate
functionally robust T cells based on a single metabolic parameter:
mitochondrial membrane potential (.DELTA..PSI.m). Mitochondria
produce energy by establishing an electrochemical proton motive
force (.DELTA.p) across their inner cell membrane, which in turn
fuels the synthesis of ATP by driving the proton turbine F0F1
ATPase. Sukumar et al. utilized a lipophilic cationic dye
tetramethylrhodamine methyl ester (TMRM) to identify and isolate
metabolically robust T cells based on their mitochondrial membrane
potential (.DELTA..PSI.m). They showed that CD8+ T cells that are
found to have low-.DELTA..PSI.m display enhanced in vivo
persistence and greater antitumor immunity relative to
high-.DELTA..PSI.m cells. Based on these findings, the authors
claimed that they have demonstrated that metabolic sorting can
complement sorting based on conventional cell surface markers in
identifying cells with the capacity for long-term survival and
ongoing effector function after adoptive transfer. They further
believed that immunometabolomic approach to cell sorting may have
important and immediate therapeutic applications in enhancing
cell-based therapies for patients with viral-associated illness,
advanced cancer, and disorders of hematopoiesis. However, sorting
of lymphocytes based on TMRM staining and flow sorting is slow and
expensive and therefore not readily amenable to clinical
application.
SUMMARY
The disclosure provides a method for isolating cells suitable for
adoptive cell therapy. The method comprises isolating
p-glycoprotein (Pgp) positive cells. The disclosure provides a
method for isolating cells suitable for adoptive cell therapy. The
method comprises obtaining a sample; optionally enriching the
sample for T cells, NK cells, stem cells, and/or mononuclear cells;
and isolating p-glycoprotein positive (Pgp-positive) including
cells selected from the group consisting of T cells, NK cells, stem
cells, and/or mononuclear cells from the enriched sample, so as to
obtain a fraction enriched in Pgp-positive cells, thereby isolating
cells suitable for adoptive cell transfer therapy. In a further
embodiment, the method comprises contacting the sample with at
least one cytotoxic drug that is a substrate of Pgp at a
concentration appropriate to kill Pgp-negative T cells, NK cells
and/or differentiated cells. In further embodiment, the at least
one cytotoxic drug is any one or more of vincristine, vinblastin,
doxorubicin, daunorubicin, taxol, paclitaxol, etoposide,
mitoxantrone, actinomycin-D, or combinations thereof. In still a
further embodiment or alternative embodiment, the method comprises
contacting the sample with at least one phototoxic compound; and
exposing the sample to a visible light source sufficient to
activate the at least one phototoxic compound so as to kill
Pgp-negative T cells. In a further embodiment, the at least one
phototoxic compound is any one or more of
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methyl
ester hydrochloride,
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl
ester hydrochloride,
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octyl
ester hydrochloride,
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid
n-butyl ester hydrochloride, 2-(6-ethyl amino-3-ethyl
imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride, or
derivatives thereof or combinations thereof. In yet another or
further embodiment, the method comprises exposing the sample to a
physical stress, such as hyperthermic conditions, that selectively
kills Pgp-negative cells. In yet another or further embodiment, the
method comprises exposing the sample to a nutritional or metabolic
stress, such as serum starvation or growth factor starvation, which
selectively kills pgp-negative cells. In still another or
alternative embodiment of any of the foregoing the method includes
isolating the Pgp-positive cells from the sample by exposing the
sample to at least one primary antibody or antibody-like moiety
specific to p-glycoprotein. In a further embodiment, the at least
one primary antibody or antibody like moiety is conjugated to at
least one fluorescent label or at least one magnetic label or
biotin. In still a further embodiment, the method includes
optionally staining the sample with at least one secondary
antibody. In a further embodiment, the at least one secondary
antibody is conjugated to at least one fluorescent label or at
least one magnetic label or biotin. In still another embodiment of
any of the foregoing, the isolating of the Pgp-positive cells from
the sample is performed by any one or more methods selected from
immunofluorescent methods, immunomagnetic methods, immunoaffinity
methods, or combinations thereof. In still another embodiment of
any of the foregoing, the isolating of the Pgp-positive cells from
the sample is performed by any one or more methods selected from
flow cytometry, magnetic activated cell sorting,
biotin-streptavidin affinity purification, or combinations thereof.
In still another embodiment of any of the foregoing, the fraction
enriched in Pgp-positive cells contains less than 50%, less than
40%, less than 30%, less than 20%, less than 10%, less than 5%, or
less than 1% Pgp-negative cells. In yet a further embodiment, the
disclosure provides that the Pgp-positive cells are genetically
modified. In yet a further embodiment, the disclosure provides that
the Pgp-positive T cells, NK cells, stem cells, and/or mononuclear
cells are further genetically modified to express at least one
chimeric antigen receptor, T cell receptor, chimeric T cell
receptor, synthetic immune receptor, TRuC.TM. or Artemis.TM. T cell
platform, so as to obtain genetically modified Pgp-positive T
cells, NK cells, stem cells, and/or mononuclear cells.
The disclosure also provides pharmaceutical compositions comprising
the Pgp-positive T cells and/or NK cells and/or stem cells of the
disclosure and at least one pharmaceutically acceptable carrier.
The disclosure also provides pharmaceutical compositions comprising
the genetically modified Pgp-positive T cells and/or NK cells
and/or stem cells and at least one pharmaceutically acceptable
carrier. The pharmaceutical compositions can be administered to a
human or other subject to treat cancer, immune disorders, or
infections.
The disclosure also provides a method for treating cancer, immune
or infectious disorders in a subject, comprising administering a
therapeutically effective amount of a composition of the disclosure
comprising a Pgp-positive T cells and/or NK cells or genetically
engineered population thereof to the subject so as to treat cancer,
immune or infectious disorders. In one embodiment, the cancer is
B-cell lymphomas, T cell lymphomas, myeloma, myelodysplastic
syndrome, skin cancer, brain tumor, breast cancer, colon cancer,
rectal cancer, esophageal cancer, anal cancer, cancer of unknown
primary site, endocrine cancer, testicular cancer, lung cancer,
hepatocellular cancer, gastric cancer, pancreatic cancer, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the
urinary tract, cancer of reproductive organs, thyroid cancer, renal
cancer, carcinoma, melanoma, head and neck cancer, brain cancer,
prostate cancer, or leukemia. In another embodiment, the method
further comprises administering a chemotherapeutic agent. In still
another embodiment, the chemotherapeutic agent is selected from
alkylating agents, alkyl sulfonates, aziridines, ethylenimines,
methylamelamines, acetogenins, a camptothecin, bryostatin,
callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin,
eleutherobin, pancratistatin, a sarcodictyin, spongistatin,
nitrogen mustards, nitrosureas, antibiotics, dynemicin,
bisphosphonates, an esperamicin, neocarzinostatin chromophore and
related chromoprotein enediyne antibiotic chromophores,
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin,
deoxydoxorubicin, epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins, anti-metabolites, folic acid analogues,
purine analogs, pyrimidine analogs, androgens, anti-adrenals, folic
acid replenisher, aceglatone, aldophosphamide glycoside,
aminolevulinic acid, eniluracil, amsacrine, bestrabucil,
bisantrene, edatraxate, defofamine, demecolcine, diaziquone,
elformithine, elliptinium acetate, an epothilone, etoglucid,
gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids,
mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,
phenamet, pirarubicin, losoxantrone, podophyllinic acid,
2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofuran,
spirogermanium, tenuazonic acid, triaziquone,
2,2',2''-trichlorotriethylamine, trichothecenes, urethane,
vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol,
pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa,
taxoids, chloranbucil, 6-thioguanine, mercaptopurine, methotrexate,
platinum analogs, vinblastine, platinum, etoposide (VP-16),
ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone,
teniposide, edatrexate, daunomycin, aminopterin, xeloda,
ibandronate, irinotecan, topoisomerase inhibitor RFS 2000;
difluoromethylornithine, retinoids, capecitabine, combretastatin,
leucovorin, oxaliplatin, lapatinib, inhibitors of PKC-alpha, Raf,
H-Ras, EGFR and VEGF-A, tyrosine kinase inhibitors, nucleoside
analogs, mTOR inhibitors, Bcl2-family inhibitors, immunomodulatory
drugs and proteasome inhibitors that reduce cell proliferation, and
pharmaceutically acceptable salts, acids or derivatives of any of
the above, or combinations thereof. In a further embodiment, the
composition and the chemotherapeutic agent are administered
sequentially or simultaneously. In another embodiment, the method
further comprises administering an anticancer agent, such as an
antibody, antibody drug conjugate, bispecific antibody, DART, a
single domain antibody, or a non-immunoglobulin antigen binding
scaffold. In another embodiment, the method further comprises
performing stem cell transplant. In another embodiment, the method
further comprises administering an anti-infective agent. In another
embodiment, the method further comprises administering an
immunosuppressive agent.
The disclosure also provides a method for isolating Pgp-negative T
cells, NK cells, and/or mononuclear cells suitable for adoptive
cell transfer therapy, comprising obtaining a sample; optionally
enriching the sample for T cells, NK cells, and/or mononuclear
cells; and depleting Pgp-positive cells from the sample, so as to
obtain a fraction enriched in Pgp-negative cells suitable for
adoptive cell transfer therapy. In one embodiment, the method
comprises depleting the Pgp-positive cells from the sample by
exposing the sample to at least one primary antibody or antibody
like moiety specific to p-glycoprotein. In a further embodiment,
the at least one primary antibody or antibody like moiety is
conjugated to at least one fluorescent label or at least one
magnetic label or biotin. The method optionally can include
staining the sample with at least one secondary antibody. In
another embodiment, the at least one secondary antibody is
conjugated to at least one fluorescent label or at least one
magnetic label or biotin. In still another embodiment, the
depleting of the Pgp-positive cells from the sample is performed by
any one or more methods selected from immunofluorescent methods,
immunomagnetic methods, or immunoaffinity methods, or combinations
thereof. In yet another embodiment, the depleting of the
Pgp-positive cells from the sample is performed by flow cytometry,
magnetic activated cell sorting, or biotin-streptavidin based cell
sorting, or combinations thereof. In a further embodiment of any of
the foregoing, the fraction enriched in Pgp-negative T cells, NK
cells, and/or mononuclear cells contains less than 50%, less than
40%, less than 30%, less than 20%, less than 10%, less than 5%, or
less than 1% Pgp-positive cells.
The disclosure also provides a pharmaceutical composition,
comprising an amount of the fraction enriched in Pgp-negative
lymphocytes, T cells or NK cells and at least one pharmaceutically
acceptable carrier.
The disclosure also provides a method of treating an infection in
an immunodeficient HIV/AIDS subject, comprising administering a
therapeutically effective amount of the composition comprising the
fraction enriched in Pgp-negative T cells and/or NK cells to the
subject so as to treat the infection. In one embodiment, the
infection is caused by a viral, bacterial, fungal or protozoan
pathogen. In a further embodiment, the infection is caused by
cytomegalovirus, adenovirus, adeno-associated virus, BK virus,
Human Herpesvirus 6, Human Herpesvirus 8, Epstein Barr virus,
influenza virus, parainfluenza virus, measles virus, mumps virus,
rhino virus, varicella virus, herpes simplex virus 1 and 2, HIV-1,
HTLV1, Mycobacterium tuberculosis, atypical mycobacteria species,
toxoplasmosis, nocardia, aspergillus, mucor, or candida.
The disclosure also provides a method for reducing
graft-versus-host disease in a subject undergoing an allogeneic
stem cell transplant, comprising administering a therapeutically
effective amount of the composition comprising the fraction
enriched in Pgp-negative T cells and/or NK cells to the subject so
as to reduce graft-versus-host disease.
The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent
from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-L shows immunofluorescence staining and sorting to enrich
for Pgp expression cells.
FIG. 2 shows a decline in cell viability following exposure to
light in both TH9402-treated compared to non-treated cells.
FIG. 3 shows that serum starvation results in a significant
enrichment of CD34.sup.+ stem cells.
FIG. 4 shows that growth factor starvation resulted in an
enrichment of CD34.sup.+ cells.
DETAILED DESCRIPTION
As used herein and in the appended claims, the singular forms "a,"
"and," and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to "a
cell" includes a plurality of such cells and reference to "the
polynucleotide" includes reference to one or more polynucleotides
and so forth.
Also, the use of "or" means "and/or" unless stated otherwise.
Similarly, "comprise," "comprises," "comprising" "include,"
"includes," and "including" are interchangeable and not intended to
be limiting.
It is to be further understood that where descriptions of various
embodiments use the term "comprising," those skilled in the art
would understand that in some specific instances, an embodiment can
be alternatively described using language "consisting essentially
of" or "consisting of."
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Allen et
al., Remington: The Science and Practice of Pharmacy 22.sup.nd ed.,
Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction
to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and
Sainsbury, Dictionary of Microbiology and Molecular Biology
3.sup.rd ed., revised ed., J. Wiley & Sons (New York, N.Y.
2006); Smith, March's Advanced Organic Chemistry Reactions,
Mechanisms and Structure 7.sup.th ed., J. Wiley & Sons (New
York, N.Y. 2013); Singleton, Dictionary of DNA and Genome
Technology 3.sup.rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green
and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold
Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012),
provide one skilled in the art with a general guide to many of the
terms used in the present application. For references on how to
prepare antibodies, see Greenfield, Antibodies A Laboratory Manual
2.sup.nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y.,
2013); Kohler and Milstein, Derivation of specific
antibody-producing tissue culture and tumor lines by cell fusion,
Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized
immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and
Riechmann et al., Reshaping human antibodies for therapy, Nature
1988 Mar. 24, 332(6162):323-7All headings and subheading provided
herein are solely for ease of reading and should not be construed
to limit the invention. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the invention, suitable methods and materials are
described below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and specific examples are illustrative only and not
intended to be limiting.
As used herein "beneficial results" may include, but are in no way
limited to, lessening or alleviating the severity of the disease
condition, preventing the disease condition from worsening, curing
the disease condition, preventing the disease condition from
developing, lowering the chances of a patient developing the
disease condition and prolonging a patient's life or life
expectancy.
"Cancer" and "cancerous" refer to or describe the physiological
condition in mammals that is typically characterized by unregulated
cell growth. Examples of cancer include, but are not limited to
B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkins
lymphomas), T cell lymphomas, myeloma, myelodysplastic syndrome,
skin cancer, brain tumor, breast cancer, colon cancer, rectal
cancer, esophageal cancer, anal cancer, cancer of unknown primary
site, endocrine cancer, testicular cancer, lung cancer,
hepatocellular cancer, gastric cancer, pancreatic cancer, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the
urinary tract, cancer of reproductive organs thyroid cancer, renal
cancer, carcinoma, melanoma, head and neck cancer, brain cancer
(e.g., glioblastoma multiforme), prostate cancer, including but not
limited to androgen-dependent prostate cancer and
androgen-independent prostate cancer, and leukemia. Other cancer
and cell proliferative disorders will be readily recognized in the
art.
"Chemotherapeutic agents" are compounds that are known to be of use
in chemotherapy for cancer. Non-limiting examples of
chemotherapeutic agents can include alkylating agents such as
thiotepa and CYTOXAN.quadrature. cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omega1I (see, e.g., Agnew, Chem. Intl.
Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A;
bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores), aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, carabicin, caminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE; vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-alpha, Raf,
H-Ras, EGFR (e.g., erlotinib (Tarceva.RTM.)) and VEGF-A that reduce
cell proliferation and pharmaceutically acceptable salts, acids or
derivatives of any of the above or combinations thereof.
"Chimeric antigen receptors" (CAR) are artificial T cell receptors
typically used as a therapy for cancer, using a technique called
adoptive cell transfer. The essential antigen-binding, signaling,
and stimulatory functions of the TCR complex have been reduced by
genetic recombination methods to a single polypeptide chain,
generally referred to as a Chimeric Antigen Receptor (CAR). See,
e.g., Eshhar, U.S. Pat. No. 7,741,465; Eshhar, U.S. Patent
Application Publication No. 2012/0093842. CARs are constructed
specifically to stimulate T cell activation and proliferation in
response to a specific antigen to which the CAR binds. Typically
"CAR-T cells" are used, which refer to T-cells that have been
engineered to containing a chimeric antigen receptor. Thus, T
lymphocytes bearing such CARs are generally referred to as CAR-T
lymphocytes. As described more fully, below, the disclosure
provides for the isolation as well as isolated Pgp-positive cells
that can be or are modified so that they express receptors that
recognize proteins that are specific to the particular form of
cancer. Such cells can be reintroduced to a subject, wherein the
cells recognize and kill cancer cells. CARs have also been used for
adoptive cell therapy of immune and infectious diseases. In
addition to CAR, native, engineered and chimeric T cell receptors
(TCR) have also been used for adoptive cell therapy.
"Disease targeted by genetically modified cells" as used herein
encompasses the targeting of any cell involved in any manner in any
disease by a genetically modified cells the hones to the disease or
a target tissue or cell type, irrespective of whether the
genetically modified cells target diseased cells or healthy cells
to effectuate a therapeutically beneficial result.
"Genetically modified cells", "redirected cells", "genetically
engineered cells" or "modified cells" as used herein refer to cells
that have been modified to express a CAR and native, engineered or
chimeric TCR. For example, a genetically modified T-lymphocyte that
expresses a CAR is a genetically modified cell (sometimes referred
to as a CAR-T cell).
"Mammal" as used herein refers to any member of the class Mammalia,
including, without limitation, humans and nonhuman primates such as
chimpanzees and other apes and monkey species; farm animals such as
cattle, sheep, pigs, goats and horses; domestic mammals such as
dogs and cats; laboratory animals including rodents such as mice,
rats and guinea pigs, and the like. The term does not denote a
particular age or sex. Thus, adult and newborn subjects, as well as
fetuses, whether male or female, are intended to be included within
the scope of this term.
"P-glycoprotein" (P-gp or Pgp) is an ATP-dependent efflux pump with
broad substrate specificity that pumps many foreign substances out
of cells. P-glycoprotein is also known as multidrug resistance
protein 1 (MDR1) or ATP-binding cassette sub-family B member 1
(ABCB1) or cluster of differentiation 243 (CD243). As used herein
and as understood by one of skill in the art, Pgp-positive is
abbreviated (Pgp.sup.+) and Pgp-negative is abbreviated
(Pgp.sup.-).
"Stem cells" are cells capable of differentiation into other cell
types and/or which retain the ability to continually replicate.
Stem cells include those having a particular, specialized function
(e.g., tissue specific cells, parenchymal cells and progenitors
thereof). Progenitor cells (i.e., "multipotent") are cells that can
give rise to different terminally differentiated cell types, and
cells that are capable of giving rise to various progenitor cells.
Cells that give rise to some or many, but not all, of the cell
types of an organism are often termed "pluripotent" stem cells,
which are able to differentiate into any cell type in the body of a
mature organism. As will be appreciated, "multipotent"
stem/progenitor cells have a more narrow differentiation potential
than do pluripotent stem cells. Another class of cells even more
primitive (i.e., uncommitted to a particular differentiation fate)
than pluripotent stem cells are the so-called "totipotent" stem
cells (e.g., fertilized oocytes, cells of embryos at the two and
four cell stages of development), which have the ability to
differentiate into any type of cell of the particular species. For
example, a single totipotent stem cell could give rise to a
complete animal, as well as to any of the myriad of cell types
found in the particular species (e.g., humans). Moreover, in the
case of lymphocytes a "stem cell lymphocyte" or "lymphocyte
progenitor" can be a lymphocyte that has not been "activated" to
target a particular antigen. Such lymphocyte progenitors retain the
ability to be "activated" to target a particular antigen. Such
lymphocyte progenitor cells are particularly useful in generating
CAR-T cells as the progenitors can be readily targeted using a
recombinant CAR and have greater replicative potential. Stem cells
and progenitor cells of the disclosure include CD34.sup.+ cells.
The term "pluripotent hematopoietic stem cell" refers to a
hematopoietic stem cell that can give rise to all blood cell
types.
"Target cell" as used herein refers to cells which are involved in
a disease and can be targeted to prevent or treat a disease
condition by genetically modified cells of the disclosure
(including but not limited to genetically modified T-cells, NK
cells, hematopoietic stem cells, pluripotent stem cells, and
embryonic stem cells). Other target cells will be apparent to those
of skill in the art and may be used in connection with alternate
embodiments of the disclosure.
The terms "T-cell" and "T-lymphocyte" are interchangeable and used
synonymously herein. Examples include but are not limited to naive
T cells ("lymphocyte progenitors"), central memory T cells,
effector memory T cells, stem memory T cells (T.sub.scm) or
combinations thereof.
Pgp positive lymphocytes of the disclosure include the
following:
(1) T cells--(a) Pgp.sup.+ T cells and subsets as defined by one or
more immunological markers, such as CD8.sup.+, CD4.sup.+,
CD62L.sup.+, CCR7.sup.+ etc.; (b) Pgp.sup.+ T cells can also
include different stages of differentiation, such as naive T cells
("lymphocyte progenitors"), central memory T cells, effector memory
T cells, memory stem T cells (T.sub.SCM) etc.; (c) Pgp.sup.+ T
cells can also include different functional subclasses, such as
Helper T cells, Cytotoxic T cells, Natural Killer T cells or
regulatory T cells etc.; and (d) Pgp.sup.+ T cells can also be
classified based on the site from which they are obtained, such as
peripheral blood, lymph nodes, spleen, bone marrow, tissue resident
lymphocytes, tumor infiltrating lymphocytes etc.; and
(2) NK Cells (Natural Killer cells)--(a) Pgp.sup.+ NK cells and
subsets as defined by one or more immunological markers, such as
CD56hi, CD56lo etc.; (b) Pgp.sup.+ NK cells can also include NK
cells at different stages of differentiation, such as naive NK
cells etc.; (c) Pgp.sup.+ NK cells can also be classified based on
the site from which they are obtained, such as peripheral blood,
lymph nodes, spleen, bone marrow, tissue resident lymphocytes,
tumor infiltrating lymphocytes etc.
Pgp negative lymphocytes (e.g., for use in patients undergoing
allogeneic stem cell transplantation to reduce the incidence of
GVHD) include the following subsets:
(1) T cells--(a) Pgp.sup.- T cells and subsets as defined by one or
more immunological markers, such as CD8.sup.+, CD4.sup.+,
CD62L.sup.+, CCR7.sup.+ etc.; (b) Pgp.sup.- T cells can also
include T cells at different stages of differentiation, such as
naive T cells ("lymphocyte progenitors"), central memory T cells,
effector memory T cells, memory stem T cells (TSCM) etc.; (c)
Pgp.sup.- T cells can also include cells belonging to different
functional subclasses, such as Helper T cells, Cytotoxic T cells,
Natural Killer T cells or regulatory T cells etc.; and (d)
Pgp.sup.- T cells can also be classified based on the site from
which they are obtained, such as peripheral blood, lymph nodes,
spleen, bone marrow, tissue resident lymphocytes, tumor
infiltrating lymphocytes etc.
The disclosure also covers methods to isolate hematopoietic stem
cells for cellular and gene therapy applications using method that
include one or more of (1) physical stress (e.g., hyperthermia),
(2) nutritional and/or metabolic stress including, but not limited
to, serum starvation and/or growth factor starvation; (3) chemical
(e.g., exposure to chemotoxic compounds); and (4) exposure to
Pgp-transported phototoxic compounds.
The disclosure also demonstrates that Pgp expression correlates
with the degree of stem-ness of a T or NK cell so that the cells
with the highest level of Pgp expression are likely to be the most
primitive (or most stem like).
"Treatment" and "treating," as used herein refer to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition, prevent the pathologic condition, pursue or
obtain beneficial results, or lower the chances of the individual
developing the condition even if the treatment is ultimately
unsuccessful. Those in need of treatment include those already with
the condition as well as those prone to have the condition or those
in whom the condition is to be prevented.
"Tumor," as used herein refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues.
As mentioned Sukumar et al. taught that robust lymphocyte were
identifiable based on low mitochondrial membrane potential. In
contrast, the present disclosure provides methods and compositions
to identify robust lymphocyte cells using markers and methods that
are easier and clinically feasible. The disclosure demonstrates
that P-glycoprotein (P-gp or Pgp) expression is a strong indicator
of lymphocyte differentiation and biological activity. Moreover,
the disclosure provides various dyes, chemotherapeutic agents,
nutritional and/or metabolic and physical stresses useful for
selecting robust lymphocyte cell types.
The cells and methods described herein use, in some cases, one or
more markers wherein at least one marker is Pgp. A molecule is a
"marker" of a desired cell type if it is found on a sufficiently
high percentage of cells of the desired cell type, and found on a
sufficiently low percentage of cells of an undesired cell type. One
can achieve a desired level of purification of the desired cell
type from a population of cells comprising both desired and
undesired cell types by selecting for cells in the population of
cells that have the marker. A marker can be displayed on, for
example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% or more of the desired cell type, and can be
displayed on fewer than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
10%, 5%, 1% or fewer of an undesired cell type.
In one embodiment, the disclosure provides Pgp.sup.+ cells,
individually or in populations. The term "isolated" or "purified"
when referring to a cell(s) of the disclosure means cells that are
substantially free of cells lacking the phenotypic marker (e.g.,
Pgp.sup.-) or vice versa. In particular embodiments, the cells are
at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 99% free of a contaminating cell types (e.g.,
Pgp.sup.+ or Pgp.sup.- cells as the case may be). In another
embodiment, the isolated cells also are substantially free of
soluble, naturally occurring molecules. A Pgp.sup.+ cell of the
disclosure, for example, can be 99%-100% purified by, for example,
flow cytometry (e.g., FACS analysis), as discussed herein.
In one embodiment, the disclosure provides an enriched population
of Pgp.sup.+ or Pgp.sup.- cells (depending upon the desired
selection criteria). An "enriched population of cells" is one
wherein a desired cell-type of the disclosure has been partially
separated from other cell types, such that the resulting population
of cells has a greater concentration of Pgp.sup.+ or Pgp.sup.-
cells than the original population of cells (they type of desired
cell with depend upon whether you are selecting "for" or "against"
Pgp expression). The enriched population of cells can have greater
than about a 1.5-fold, 2-fold, 10-fold, 100-fold, 500-fold,
1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold,
6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold or
greater concentration of, e.g., Pgp.sup.+ cells than the original
population had prior to separation. Pgp.sup.+ cells of the
disclosure can, for example, make up at least 5%, 10%, 15%, 20%,
35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 99% or more of the enriched population of stem cells. The
enriched population of cells may be obtained by, for example,
selecting against cells displaying lacking a Pgp marker.
Alternatively, or in addition to, the enrichment for the expression
of a marker, the loss of expression of a marker may also be used
for enrichment. For example, lack of expression of a marker (e.g.,
Pgp) can be used to select cells.
In another embodiment, the disclosure uses chemotherapeutic agents
to select robust lymphocyte cells. As mentioned above, Pgp is a
multidrug resistance protein that pumps a broad range of
substrates, including harmfuls substrates such as
chemotherapeutics, out of cells. Thus, cells that express Pgp
including high levels of Pgp are more resistant to
chemotherapeutics. Accordingly, contacting a population of cells
expressing various levels of Pgp with a chemotherapeutic(s) will
select for those cells that have higher expression levels of Pgp
compared to those with no or low level expression of Pgp.
Similarly, dyes (e.g., photosensitive dyes) can be used in a
similar fashion. In this embodiment, cells that express or over
express Pgp will tend to pump out (or exclude) the dye compared to
cells with low or no Pgp expression. Dye-containing cells can be
separate from non-dye-containing cells. Moreover, if the dye is
toxic or can be photoactivatated, cells that contain the dye can be
killed thus retaining the Pgp expressing cells.
The disclosure also demonstrates that exposing cells to physical
stresses, such as hyperthermic conditions, can provide selection.
For example, exposing lymphocytes to higher temperatures for short
periods of time provides a selective advantage to cells that
express or over express Pgp compared to cells that do no expression
Pgp.
The disclosure also demonstrates that exposing cells to
serum-starvation and growth factor-starvation conditions can
provide enrichment. For example, exposing peripheral blood stem
cells to serum starvation for short periods of time results in
enrichment of CD34+ cells that express or coexpress Pgp.
As disclosed herein, T cells expressing Pgp have better capacity
for long-term survival and ongoing effector function after adoptive
transfer. Thus, in one embodiment, expression and/or activity of
P-glycoprotein can be used to select lymphocytes for adoptive
cellular therapy including, but not limited to, genetic
modification by CAR to produce CAR-T cells, TCRs and chimeric TCRs.
Since P-glycoprotein is a cell surface protein and there a number
of antibodies available against it, the current disclosure provides
a method of selecting lymphocytes for adoptive cell therapy based
on P-glycoprotein expression. The P-glycoprotein positive cells of
the disclosure can be isolated using a number of techniques known
in the art, such as magnetic activated cell sorting (MACS), in
addition to conventional flow sorting. These techniques have the
advantage over the method proposed by Sukumar et al. in that they
are quick, economical and amenable to clinical application as a
number of systems for MACS are already in clinical use. For
example, Miltenyi Biotech markets a system for MACS that has FDA
approval and is in clinical use in several centers.
P-glycoprotein expression can also be combined with positive and
negative selection of other markers (e.g., CD8, CD4, CD62L, CD44,
etc.) to further enrich lymphocytes with stem-like property and/or
to enrich for stem-like T cells belonging to different subsets
(e.g. cytotoxic, helper, Treg, etc.) for adoptive cellular
therapy.
Provided herein are methods for isolating cells suitable for
adoptive cell therapy. In one embodiment the methods comprise
obtaining a sample, enriching the sample for T cells, mononuclear
cells, NK cells, and/or stem cells and isolating p-glycoprotein
positive (Pgp.sup.+) T cells, mononuclear cells, NK cells and/or
stem cells from the enriched sample, so as to obtain a fraction
enriched in Pgp.sup.+ T cells, NK cells, and/or stem cells suitable
for adoptive cell transfer therapy. In some embodiments, the step
of enriching the sample for T cells, mononuclear cells, NK cells
and/or stem cells can be omitted. In some embodiments, isolating
the Pgp.sup.+ T cells, NK cells and/or stem cells from the sample
comprises exposing the sample to at least one primary antibody or
antibody-like moiety specific to p-glycoprotein. In some
embodiments, the at least one primary antibody or antibody-like
moiety is conjugated to at least one fluorescent label or at least
one magnetic label. In some embodiments, the methods further
comprise optionally staining the sample with at least one secondary
antibody. In some embodiments, the at least one secondary antibody
is conjugated to at least one fluorescent label or at least one
magnetic label. In some embodiments, isolating of the Pgp.sup.+ T
cells, NK cells and/or stem cells from the sample is performed by
any one or more methods selected from immunofluorescent methods,
immunomagnetic methods, immunoaffinity methods or combinations
thereof. In some embodiments, isolating of the Pgp.sup.+ T cells,
NK cells and/or stem cells from the sample is performed by any one
or more methods selected from flow cytometry, magnetic activated
cell sorting, biotin-streptavidin based affinity purification or
combinations thereof. In some embodiments, Pgp.sup.+ T cells, NK
cells and/or stem cells can be isolated from a sample (e.g. blood,
bone marrow, leukopheresis sample, or peripheral blood mononuclear
cells) in a single step by simultaneous labeling with
fluorochrome-conjugated antibodies against Pgp and other cellular
markers associated with the cell type (e.g., T cell marker(s) such
as CD3) followed by sorting for Pgp+/marker.sup.2 (e.g., followed
by sorting for Pgp.sup.+/CD3.sup.+ T cells) by flow sorting. In
some embodiments, Pgp.sup.+ T cell subsets can be isolated from a
sample (e.g. blood, bone marrow, leukopheresis sample, or
peripheral blood mononuclear cells) in a single step by
simultaneous labeling with fluorochrome-conjugated antibodies
against Pgp and T cell subset marker(s) (e.g. CD4, CD8, etc.)
followed by sorting for Pgp.sup.+/CD4.sup.+ or Pgp.sup.+/CD8.sup.+
T cells by flow sorting.
In addition to separation of Pgp expressing cells based on surface
labeling with Pgp antibodies, the disclosure provides methods of
isolation/purification/enrichment of lymphocytes for adoptive
cellular therapy based on Pgp activity. A number of cytotoxic
drugs, such as Vincristine, vinnblastin, doxorubicin, daunorubicin,
taxol, paclitaxol, etoposide, mitoxantrone, actinomycin-D, etc. are
substrates of Pgp. Therefore, Pgp-expressing cells can be enriched
by exposing T cells to appropriate concentration of the above drugs
that will kill Pgp-negative cells, thus
isolating/purifying/enriching Pgp.sup.+ cells.
In another embodiment, the disclosure provides a method for
isolating cells suitable for adoptive cell therapy, comprising
obtaining a sample, enriching the sample for T cells, contacting
the sample with at least one cytotoxic drug at a concentration
appropriate to kill Pgp.sup.- T cells, and isolating Pgp.sup.+ T
cells from the enriched sample, so as to isolate cells suitable for
adoptive cell transfer therapy. In another embodiment, the
disclosure provides a method for isolating cells suitable for
adoptive cell therapy, comprising obtaining a sample, enriching the
sample for mononuclear cells, contacting the sample with at least
one cytotoxic drug at a concentration appropriate to kill Pgp.sup.-
mononuclear cells, and isolating Pgp.sup.+ mononuclear cells from
the enriched sample, so as to isolate cells suitable for adoptive
cell transfer therapy. In some embodiments, the step of enriching
the sample for T cells or mononuclear cells can be omitted prior to
contain the cells with a cytotoxic drug. In some embodiments, the
cytotoxic drugs are any one or more of vincristine, vinnblastin,
doxorubicin, or taxol, or combinations thereof. In any of the
foregoing embodiments, the cell population enriched for Pgp.sup.+
cells can be further processed using antibodies to markers on the
cells (e.g., anti-Pgp antibodies), dyes, or hyperthermic
conditions. In some embodiments, isolating the Pgp.sup.+ T cells
from the sample comprises exposing the sample to at least one
primary antibody or antibody like moiety specific to
p-glycoprotein. In some embodiments, the at least one primary
antibody or antibody like moiety is conjugated to at least one
fluorescent label or at least one magnetic label. In some
embodiments, the methods further comprise optionally staining the
sample with at least one secondary antibody. In some embodiments,
the at least one secondary antibody is conjugated to at least one
fluorescent label or at least one magnetic label. In some
embodiments, isolating of the Pgp.sup.+ T cells from the sample is
performed by any one or more methods selected from
immunofluorescent methods, immunomagnetic methods, or combinations
thereof. In some embodiments, isolating of the Pgp.sup.- positive T
cells from the sample is performed by any one or more methods
selected from flow cytometry, magnetic activated cell sorting, or
combinations thereof.
In addition, Pgp expressing cells can be purified/enriched/isolated
using photodynamic dye treatment. For example, Pgp expression cells
can be purified/enriched/isolated with rhodamine analogs that are
differentially retained between Pgp.sup.+ and Pgp.sup.- cells and
become phototoxic on exposure to visible light. This method can be
used alone or in combination with other methods of selection
described herein. Exemplary rhodamine and rhodamine derivatives are
known and include, for example, derivatives selected from the group
consisting of 4,5-dibromorhodamine 123
(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid
methyl ester hydrochloride); 4,5-dibromorhodamine 123
(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl
ester hydrochloride); 4,5-dibromorhodamine 123
(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octyl
ester hydrochloride); 4,5-dibromorhodamine 110 n-butyl ester
(2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid
n-butyl ester hydrochloride); Rhodamine B n-butyl ester (2-(6-ethyl
amino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester
hydrochloride); and photoactivable derivatives thereof; whereby
photoactivation of said derivatives induces cell killing while
unactivated derivatives are substantially non-toxic to cells. Other
phototoxic compounds that are substrate of Pgp have been described
in the literature as well and can be used in the method of the
current disclosure. For example, using a rhodamine or rhodamine
analog, a population of cells can be contacted with a rhodamine or
rhodamine analog under sufficient concentrations and time that
allow for cells to take up the rhodamine or rhodamine analog. As
described herein and above, Pgp expressing cells will pump the
rhodamine or rhodamine analog out of the cell, while Pgp.sup.- or
cells having reduced Pgp expression will retain the rhodamine or
rhodamine analog. The population is then contacted with a light of
a wavelength that causes ablation of cells containing the rhodamine
or rhodamine analog (i.e., cells lacking or having reduced Pgp
expression). Thus, the population will be enriched for Pgp.sup.+
cells by photo-ablation of Pgp.sup.- cells.
Due to the specific retention of the rhodamine 123 (Rh123) class of
dyes by Pgp.sup.- cells and the concomitant lack of their
accumulation by the lymphocytes with stem-like phenotype (e.g., by
Pgp.sup.+), the disclosure provides a method for the use of these
dyes for in vivo or in vitro photodynamic therapy to enrich
lymphocytes for adoptive cell therapy.
Since low staining with TMRM, Rh123 and DiOC2(3) correlates with
and/or is primarily due to Pgp mediated efflux and is not solely
due to low mitochondrial membrane potential, the disclosure teaches
an optimized protocol for isolation of Pgp expressing cells by
optimizing Pgp mediated efflux. For example, by performing the
assay at 37.degree. C. and by allowing more time for Pgp mediated
efflux of the dyes, a better differentiation can be obtained
between Pgp.sup.+ and Pgp.sup.- cells for the purpose of adoptive
cellular therapy.
In another embodiment, the disclosure provides a method for
isolating cells suitable for adoptive cell therapy, comprising
obtaining a sample, enriching the sample for T cells, NK cells,
stem cells, and/or mononuclear cells, contacting the sample with at
least one phototoxic compound, exposing the sample to a visible
light source sufficient to activate the at least one phototoxic
compound so as to kill Pgp.sup.- T cells, NK cells, stem cells,
and/or mononuclear cells, and isolating Pgp.sup.+ T cells, NK
cells, stem cells, and/or mononuclear cells from the enriched
sample, so as to isolate cells suitable for adoptive cell transfer
therapy. This method can be used alone or in combination with other
methods for Pgp.sup.+ cell enrichment. In some embodiments, the
step of enriching the sample for T cells, NK cells, stem cells,
and/or mononuclear cells can be omitted. In some embodiments, the
phototoxic compounds are any one or more of
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methyl
ester hydrochloride,
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl
ester hydrochloride,
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octyl
ester hydrochloride,
2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid
n-butyl ester hydrochloride, 2-(6-ethyl amino-3-ethyl
imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride, or
derivatives thereof or combinations thereof. For example, using a
phototoxic, a population of cells can be contacted with a
phototoxic compound under sufficient concentrations and time that
allow for cells to take up the phototoxic compound. As described
herein and above, Pgp expressing cells will pump the phototoxic
compound out of the cell, while Pgp.sup.- or cells having reduced
Pgp expression will retain the phototoxic compound. The population
is then contacted with a light of a wavelength that causes ablation
of cells containing the phototoxic compound (i.e., cells lacking or
having reduced Pgp expression). Thus, the population will be
enriched for Pgp.sup.+ cells by photo-ablation of Pgp.sup.-
cells.
In another embodiment, physical methods, such as temperature
exposure, can be used to select for CD34.sup.+ and/or Pgp.sup.+
cells. In this embodiment, the disclosure provides a method for
isolating cells suitable for adoptive cell therapy, comprising
obtaining a sample, enriching the sample for T cells, NK cells,
stem cells, and/or mononuclear cells, culturing the cells and
exposing the population of cells to hyperthermia so as to kill
Pgp.sup.- T cells, NK cells, stem cells, and/or mononuclear cells,
and isolating/enriching for Pgp.sup.+ T cells, NK cells, stem
cells, and/or mononuclear cells. In one embodiment, the cells are
exposed to a temperature from about 40-42.degree. C. for 2-4 hours.
In one embodiment, the temperature is 42-43.degree. C. In another
embodiment, the temperature is 42.degree. C. for 2-3 hours. In
another embodiment, the temperature is 43.degree. C. for 2-3 hours.
This method can be used alone or in combination with other methods
for Pgp.sup.+ cell enrichment. In some embodiments, the step of
enriching the sample for T cells, NK cells, stem cells, and/or
mononuclear cells can be omitted. For example, using a hyperthermic
temperature during culturing, a population of cells can be enriched
for Pgp.sup.+ cells.
In another embodiment, temperature exposure can be used to select
for Pgp.sup.+/CD34.sup.+ cells. In this embodiment, the disclosure
provides a method for isolating cells suitable for adoptive cell
therapy, comprising obtaining a sample, enriching the sample for T
cells or mononuclear cells, culturing the cells and exposing the
population of cells to hyperthermia so as to kill Pgp.sup.- T, NK
or mononuclear cells, and isolating/enriching for Pgp.sup.+ stem
cells and mononuclear cells. In one embodiment, the cells are
exposed to a temperature from about 40-42.degree. C. for 2-4 hours.
In one embodiment, the temperature is 42-43.degree. C. In another
embodiment, the temperature is 42.degree. C. for 2-3 hours. In
another embodiment, the temperature is 43.degree. C. for 2-3 hours.
The cells can then be "panned" for CD34.sup.+ marker. This method
can be used alone or in combination with other methods for
Pgp.sup.+/CD34.sup.+ cell enrichment. For example, using a
hyperthermic temperature during culturing, a population of cells
can be enriched for Pgp.sup.+/CD34.sup.+ cells.
In some embodiments, where cytotoxic agents are used (e.g.,
chemotherapeutics and/or photoactive dyes and/or other agents)
and/or physical stress (such as hyperthermic treatment) and/or
nutritional/metabolic stress (e.g., serum- or growth factor
starvation/depletion) are used for isolating the Pgp.sup.+ cells
from the sample the method can further include, after or prior to
the above methods, exposing the sample to at least one primary
antibody or antibody like moiety specific to p-glycoprotein. In
some embodiments, the at least one primary antibody or antibody
like moiety is conjugated to at least one fluorescent label or at
least one magnetic label or biotin. In some embodiments, the
methods further comprise optionally staining the sample with at
least one secondary antibody. In some embodiments, the at least one
secondary antibody is conjugated to at least one fluorescent label
or at least one magnetic label or biotin. In some embodiments,
isolating of the Pgp.sup.+ T cells from the sample is performed by
any one or more methods selected from immunofluorescent methods,
immunomagnetic methods, immunoaffinity methods or combinations
thereof. In some embodiments, isolating of the Pgp.sup.+ T cells
from the sample is performed by any one or more methods selected
from flow cytometry, magnetic activated cell sorting,
biotin-streptavidin based cell sorting or combinations thereof.
Although not necessary, but using a combination of purification
methods the enrichment of Pgp.sup.+ cells can be further improved
and/or optimized. In some embodiments, the fraction enriched in
Pgp.sup.+ T cells contains less than 50% Pgp.sup.- T cells. In some
embodiments, the fraction enriched in Pgp.sup.+ T cells contains
less than 40% Pgp.sup.- T cells. In some embodiments, the fraction
enriched in Pgp.sup.+ T cells contains less than 30% Pgp.sup.- T
cells. In some embodiments, the fraction enriched in Pgp.sup.+ T
cells contains less than 20% Pgp.sup.- T cells. In some
embodiments, the fraction enriched in Pgp.sup.+ T cells contains
less than 10% Pgp.sup.- T cells. In some embodiments, the fraction
enriched in Pgp.sup.+ T cells contains less than 5% Pgp.sup.- T
cells. In some embodiments, the fraction enriched in Pgp.sup.+ T
cells contains less than 1% Pgp.sup.- T cells.
In some embodiments, the fraction enriched in Pgp.sup.+ mononuclear
cells contains less than 50% Pgp.sup.- mononuclear cells. In some
embodiments, the fraction enriched in Pgp.sup.+ mononuclear cells
contains less than 40% Pgp.sup.- mononuclear cells. In some
embodiments, the fraction enriched in Pgp.sup.+ mononuclear cells
contains less than 30% Pgp.sup.- mononuclear cells. In some
embodiments, the fraction enriched in Pgp.sup.+ mononuclear cells
contains less than 20% Pgp.sup.- mononuclear cells. In some
embodiments, the fraction enriched in Pgp.sup.+ mononuclear cells
contains less than 10% Pgp.sup.- mononuclear cells. In some
embodiments, the fraction enriched in Pgp.sup.+ mononuclear cells
contains less than 5% Pgp.sup.- mononuclear cells. In some
embodiments, the fraction enriched in Pgp.sup.+ mononuclear cells
contains less than 1% Pgp.sup.- mononuclear cells.
In addition to Pgp, a number of other drug transporters (including
breast cancer resistance protein) are selectively expressed on the
surface of stem cells. Antibodies and substrates of these proteins
can be also used to enrich lymphocytes with stem like phenotype for
the purpose of adoptive cellular therapies. Thus, using selection
techniques for these markers in combination with the selection
techniques described herein for Pgp cell can improve cell-selection
processing techniques.
Once the Pgp expressing cells have been enriched by any of the
above methods, the cells can be used for gene modification with
CAR, TCR, chimeric TCR, synthetic immune receptor, TRuC.TM. T cell
platform, Artemis.TM. T cell platform or other methods for the
purpose of adoptive cellular therapy. They could be also used
without gene modification, for example for immunization with T cell
antigens. In some embodiments, the Pgp.sup.+ T cells obtained by
the methods described herein are genetically modified for use in
adoptive cell therapy. In exemplary embodiments, the Pgp.sup.+ T
cells are genetically modified to express at least one chimeric
antigen receptor, T cell receptor, chimeric T cell receptor,
synthetic immune receptor, TRuC.TM. T cell platform, Artemis.TM. T
cell platform for therapeutic uses, such as for treating
cancer.
In some embodiments, the genetically modified Pgp.sup.+ T cells
according to any of the methods disclosed herein may be used in
treating cancer, infection or immune disorders. Accordingly, in
various embodiments, the disclosure provides methods for treating
cancer or immune disorders in a subject, comprising providing
genetically modified Pgp.sup.+ cells described herein, and
administering a therapeutically effective amount of the cells to
the subject so as to treat cancer.
In some embodiments, the cancer is B-cell lymphomas, T cell
lymphoma, skin cancer, testicular cancer, endocrine cancer, cancer
of unknown primary site, rectal cancer, anal cancer, esophageal
cancer, brain tumor (e.g., glioblastoma multiforme), breast cancer,
colon cancer, lung cancer, hepatocellular cancer, gastric cancer,
pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, cancer of the urinary tract, cancer of reproductive
tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and
neck cancer, brain cancer, prostate cancer, or leukemia. In some
embodiments, the cancer is B-cell lymphomas, T cell lymphomas,
myeloma, myelodysplastic syndrome, skin cancer, brain tumor, breast
cancer, colon cancer, rectal cancer, esophageal cancer, anal
cancer, cancer of unknown primary site, endocrine cancer,
testicular cancer, lung cancer, hepatocellular cancer, gastric
cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, cancer of the urinary tract, cancer of
reproductive organs, thyroid cancer, renal cancer, carcinoma,
melanoma, head and neck cancer, brain cancer, prostate cancer, or
leukemia.
In some embodiments, the genetically modified Pgp.sup.+ cells
described herein are administered simultaneously or sequentially
with a chemotherapeutic agent. In some embodiments, the
chemotherapeutic agent is selected from alkylating agents, alkyl
sulfonates, aziridines, ethylenimines, methylamelamines,
acetogenins, a camptothecin, bryostatin, callystatin, CC-1065,
cryptophycins, dolastatin, duocarmycin, eleutherobin,
pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards,
nitrosureas, antibiotics, dynemicin, bisphosphonates, an
esperamicin, neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores, aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, anti-metabolites, folic acid
analogues, purine analogs, pyrimidine analogs, androgens,
anti-adrenals, folic acid replenisher, aceglatone, aldophosphamide
glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil,
bisantrene, edatraxate, defofamine, demecolcine, diaziquone,
elformithine, elliptinium acetate, an epothilone, etoglucid,
gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids,
mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,
phenamet, pirarubicin, losoxantrone, podophyllinic acid,
2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofuran,
spirogermanium, tenuazonic acid, triaziquone,
2,2',2''-trichlorotriethylamine, trichothecenes, urethane,
vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol,
pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa,
taxoids, chloranbucil, 6-thioguanine, mercaptopurine, methotrexate,
platinum analogs, vinblastine, platinum, etoposide (VP-16),
ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone,
teniposide, edatrexate, daunomycin, aminopterin, xeloda,
ibandronate, irinotecan, topoisomerase inhibitor RFS 2000;
difluoromethylornithine, retinoids, capecitabine, combretastatin,
leucovorin, oxaliplatin, lapatinib, inhibitors of PKC-alpha, Raf,
H-Ras, EGFR and VEGF-A that reduce cell proliferation, and
pharmaceutically acceptable salts, acids or derivatives of any of
the above, or combinations thereof.
In addition to enriching Pgp-expressing immune cells for the
purpose of adoptive T cell therapies, the disclosure also teaches
methods to deplete Pgp-expressing immune cells from stem cell
grafts given to patients undergoing allogeneic stem cell transplant
to lower the risk of Graft versus Host disease. The disclosure also
teaches methods to deplete Pgp-expressing immune cells from donor T
cell given to immunodeficient patients to boost their immunity
against infections while lowering the risk of Graft-vs-Host Disease
(GVHD). One of skill in the art would recognize that methods
described herein for killing or ablation of Pgp.sup.- cells (i.e.,
the selective killing of Pgp.sup.-) would not be useful for
depleting a sample of Pgp.sup.+ cells. Rather methods that are
capable of selectively killing or removing Pgp.sup.+ cells while
leaving the Pgp.sup.- cells intact would be used.
Graft-versus-host disease (GVHD) is the main cause of mortality and
a major limitation to the early and widespread use of allogeneic
stem cell transplantation (SCT), a treatment that often represents
the only curative option for numerous patients with malignant
diseases and hereditary metabolic disorders. Depletion of T cells
capable of recognizing and mounting an immune response toward host
cells from stem cell grafts can reduce or even eliminate GVHD.
However, the elimination of T cells also results in delayed T-cell
reconstitution and, thus, an increased rate of infection,
particularly with viral agents such as cytomegalovirus, herpes
zoster, and Epstein-Barr virus. In addition, the eradication of
mature T cells is associated with an increased risk of graft
rejection and an increased incidence of relapse of malignant
disease. Thus, T cells are required early after allogeneic
transplantation and depleting the graft of its T-cell content is
not an ideal approach to prevention of complications after
transplantation. Although new immunosuppressive agents offer
options to decrease the incidence and severity of GVHD, most of the
time these strategies are only partially effective and may also
increase the incidence of viral and fungal infections and other
adverse effects of profound immunosuppression.
To provide a solution to this problem, selective inactivation or
elimination of alloreactive donor T lymphocytes could allow early
immune recovery and response toward infectious agents, and
potentially preserve graft-versus-leukemia (GVL) activity. In
addition, a strategy to selectively eliminate immunoreactive T
cells could represent an important advance for the treatment of a
large number of patients with autoimmune disorders. In this
procedure, stimulation of T cells with mitogens or allogeneic major
histocompatibility complex-mismatched cells resulted in the
preferential retention of the TH9402 rhodamine-derivative in
activated T cells, both CD4 and CD8. Photodynamic cell therapy of
TH9402-exposed T cells led to the selective elimination of
immunoreactive T-cell populations.
Based on the discovery that Pgp is expressed on T stem cells and
its expression is lost upon T cell activation, Pgp.sup.- cells in a
donor have been already exposed to an antigen. The likely antigens
for such Pgp.sup.- cells in a healthy donor are likely to be common
pathogens, such as viruses and fungi. Thus, Pgp.sup.- cells may
confer immunity to pathogens commonly encountered in the
environment. In contrast, Pgp.sup.+ cells are likely to contain
naive cells that can cause GVHD when given to a donor. Therefore,
elimination of Pgp.sup.+ cells from a graft may enrich for T cells
that can confer immunity while sparing GVHD. In the present
disclosure the method depletes Pgp-expressing T cells obtained from
a donor. Thus, the present method involves depletion of Pgp.sup.+
cells. In contrast to other methods, the method of present
disclosure does not involve stimulation of donor T cells with
mitogen or MHC mismatched host cells. Finally, the method of the
present disclosure involves depletion of Pgp expressing cells by
staining with Pgp antibody or an antibody like moiety (e.g. scFv,
vHH, affibody, nanobody, Fab fragment, Darpins etc.). In the method
described herein, the depletion of Pgp expressing cells is achieved
by staining T cells with more than 1 antibody or antibody like
moieties directed against different epitopes on the extracellular
domain of Pgp. Since Pgp is also expressed on normal hematopoietic
stem cells, depletion of Pgp expressing cells would potentially
deplete stem cells from the graft. As such, in one method of the
disclosure, stem cells are first positively selected from the graft
using an antibody against CD34 antigen. This can be achieved using
a commercially available CD34 isolation system (Miltenyi).
Subsequently, the CD34-negative flow through fraction of the graft
is depleted of Pgp expressing cells by immunostaining with Pgp
antibody or a cocktail of antibodies. The CD34.sup.+ stem cell
fraction is then administered with CD34.sup.-/Pgp.sup.- T cell
fraction to the patient undergoing allogeneic bone marrow,
peripheral blood stem cell, or umbilical cord stem cell transplant.
The Pgp.sup.- T cell fraction can also be given to the patient at a
later time than CD34.sup.+ stem cell infusion.
Another application of the disclosure is for adoptive cellular
therapy in immunodeficient patients, such as allogeneic stem cell
transplant (including umbilical cord transplant) recipients, who
are immunodeficient and have become infected due to poor T cell
reconstitution. Administration of a T cell population that is
enriched for T cells capable of conferring immunity to viral,
bacterial and fungal pathogens, but have limited capacity for
alloreactivity or to cause GVHD, is highly desirable in this
setting. Therefore, administration of Pgp.sup.- T cell population
obtained from the donor (primary donor or even third party donors)
to such patients will protect against infectious agents while not
significantly increasing the risk of GVHD.
In one embodiment, the disclosure provides a method for isolating
Pgp.sup.- T cells suitable for adoptive cell transfer therapy,
comprising obtaining a sample, enriching the sample for T cells,
and depleting Pgp.sup.+ T cells from the sample, so as to obtain a
fraction enriched in Pgp.sup.- T cells suitable for adoptive cell
transfer therapy. In some embodiments, depleting the Pgp.sup.+ T
cells from the sample comprises exposing the sample to at least one
primary antibody or antibody like moiety specific to
p-glycoprotein. In some embodiments, the at least one primary
antibody or antibody like moiety is conjugated to at least one
fluorescent label or at least one magnetic label or biotin. In some
embodiments, the methods further comprise optionally staining the
sample with at least one secondary antibody. In some embodiments,
the at least one secondary antibody is conjugated to at least one
fluorescent label or at least one magnetic label or biotin. In some
embodiments, depleting of the Pgp.sup.+ T cells from the sample is
performed by any one or more methods selected from
immunofluorescent methods, immunomagnetic methods, immunoaffinity
methods or combinations thereof. In some embodiments, depleting of
the Pgp.sup.+ T cells from the sample is performed by flow
cytometry, magnetic activated cell sorting, Biotin-streptavidin
based immunoaffinity cell sorting or combinations thereof. In some
embodiments, the fraction enriched in Pgp.sup.- T cells contains
less than 50% Pgp.sup.+ T cells. In some embodiments, the fraction
enriched in Pgp-negative T cells contains less than 40% Pgp.sup.+ T
cells. In some embodiments, the fraction enriched in Pgp.sup.- T
cells contains less than 30% Pgp.sup.+ T cells. In some
embodiments, the fraction enriched in Pgp.sup.- T cells contains
less than 20% Pgp.sup.+ T cells. In some embodiments, the fraction
enriched in Pgp.sup.- T cells contains less than 10% Pgp.sup.+ T
cells. In some embodiments, the fraction enriched in Pgp.sup.- T
cells contains less than 5% Pgp.sup.+ T cells. In some embodiments,
the fraction enriched in Pgp.sup.- T cells contains less than 1%
Pgp.sup.+ T cells.
In some embodiments, the disclosure provides a method for the
treatment of infection in an immunodeficient HIV/AIDS subject,
comprising providing a composition comprising a population of
Pgp.sup.- T cells isolated by the methods described herein and
administering a therapeutically effective amount of the composition
to the subject so as to treat the infection. The infection may be
viral infection (e.g. cytomegalovirus, adenovirus or BK virus), a
bacterial infection (e.g. mycobacterium, enterococcus), fungal
infection (e.g. mucor or aspergillus) or protozoan infection (e.g.
toxoplasmosis).
In some embodiments, the disclosure provides a method for the
treatment of infection in a subject undergoing an allogeneic stem
cell transplant, comprising providing a composition comprising a
population of Pgp.sup.- T cells isolated by the methods described
herein and administering a therapeutically effective amount of the
composition comprising the Pgp.sup.- cells to the subject so as to
treat the infection. The infection may be viral infection (e.g.
cytomegalovirus, adenovirus or BK virus), a bacterial infection
(e.g. mycobacterium, enterococcus), fungal infection (e.g. mucor or
aspergillus) or protozoan infection (e.g. toxoplasmosis).
In some embodiments, the disclosure provides a method for reducing
graft-versus-host disease in a subject undergoing an allogeneic
stem cell transplant, comprising providing a composition comprising
a population of Pgp.sup.- T cells isolated by the methods described
herein and administering a therapeutically effective amount of the
composition comprising the Pgp.sup.+ T cells to the subject so as
to reduce graft-versus-host disease. In some embodiments, the
transplant is an allogeneic bone marrow transplant, an allogeneic
peripheral blood stem cell transplant, a haploidentical bone marrow
transplant, a haploidentical peripheral blood stem cell transplant,
or an umbilical cord stem cell transplant.
In various embodiments, the disclosure provides pharmaceutical
compositions comprising a population of the Pgp.sup.+ T cells
isolated by any of the methods disclosed herein, and at least one
pharmaceutically acceptable carrier. In some embodiments, the
Pgp.sup.+ T cells are genetically modified as described herein. In
some embodiments, the pharmaceutical compositions comprising the
genetically modified Pgp.sup.+ T cells are used in cancer therapies
as described herein. In some embodiments, the pharmaceutical
compositions comprising the genetically modified Pgp.sup.+ T cells
are used in the treatment of infectious and immune disorders as
described herein.
In various embodiments, the disclosure provides pharmaceutical
compositions comprising a population of Pgp.sup.- T cells isolated
by any of the methods described herein, and at least one
pharmaceutically acceptable carrier. In various embodiments, the
pharmaceutical compositions comprising the Pgp.sup.- T cells
isolated by the methods described herein are used in therapies
including but not limited to reduction of graft-versus-host disease
in a subject undergoing allogeneic transplant (including stem cell
transplant), treatment of an infection in a subject in need thereof
and/or treatment of an infection in a HIV/AIDS subject, wherein the
infection may be viral infection (e.g. cytomegalovirus, adenovirus
or BK virus), a bacterial infection (e.g. mycobacterium,
enterococcus), fungal infection (e.g. mucor or aspergillus) or
protozoan infection (e.g. toxoplasmosis).
The pharmaceutical compositions according to the invention can
contain any pharmaceutically acceptable excipient.
"Pharmaceutically acceptable excipient" means an excipient that is
useful in preparing a pharmaceutical composition that is generally
safe, non-toxic, and desirable, and includes excipients that are
acceptable for veterinary use as well as for human pharmaceutical
use. Such excipients may be solid, liquid, semisolid, or, in the
case of an aerosol composition, gaseous. Examples of excipients
include but are not limited to starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents, wetting agents, emulsifiers, coloring
agents, release agents, coating agents, sweetening agents,
flavoring agents, perfuming agents, preservatives, antioxidants,
plasticizers, gelling agents, thickeners, hardeners, setting
agents, suspending agents, surfactants, humectants, carriers,
stabilizers, and combinations thereof.
In various embodiments, the pharmaceutical compositions according
to the disclosure may be formulated for delivery via any route of
administration. "Route of administration" may refer to any
administration pathway known in the art, including but not limited
to aerosol, nasal, oral, transmucosal, transdermal, parenteral or
enteral. "Parenteral" refers to a route of administration that is
generally associated with injection, including intraorbital,
infusion, intraarterial, intracapsular, intracardiac, intradermal,
intramuscular, intraperitoneal, intrapulmonary, intraspinal,
intrasternal, intrathecal, intrauterine, intravenous, subarachnoid,
subcapsular, subcutaneous, transmucosal, or transtracheal. Via the
parenteral route, the compositions may be in the form of solutions
or suspensions for infusion or for injection. Via the parenteral
route, the compositions may be in the form of solutions or
suspensions for infusion or for injection. Via the enteral route,
the pharmaceutical compositions can be in the form of gel capsules,
syrups, suspensions, solutions, emulsions, microspheres or lipid
vesicles or polymer vesicles. Typically, the compositions are
administered by injection. Methods for these administrations are
known to one skilled in the art. In another embodiment, the
compositions can be part of a tissue delivery device or implant. In
such embodiments, the cells are allowed to grow and/or exist in a
biocompatible implantable structure (e.g., with in collagen matrix
and the like). In another embodiment, the cells may be applied to a
structure prior to implantation (e.g., a stent, balloon, valve,
pump etc.).
The pharmaceutical compositions according to the disclosure can
contain any pharmaceutically acceptable carrier. "Pharmaceutically
acceptable carrier" as used herein refers to a pharmaceutically
acceptable material, composition, or vehicle that is involved in
carrying or transporting a compound of interest from one tissue,
organ, or portion of the body to another tissue, organ, or portion
of the body. For example, the carrier may be a liquid or solid
filler, diluent, excipient, solvent, or encapsulating material, or
a combination thereof. Each component of the carrier must be
"pharmaceutically acceptable" in that it must be compatible with
the other ingredients of the formulation. It must also be suitable
for use in contact with any tissues or organs with which it may
come in contact, meaning that it must not carry a risk of toxicity,
irritation, allergic response, immunogenicity, or any other
complication that excessively outweighs its therapeutic
benefits.
The pharmaceutical compositions according to the disclosure can
also be encapsulated or prepared in an emulsion or syrup for oral
administration. Pharmaceutically acceptable solid or liquid
carriers may be added to enhance or stabilize the composition, or
to facilitate preparation of the composition. Liquid carriers
include syrup, peanut oil, olive oil, glycerin, saline, alcohols
and water. Solid carriers include starch, lactose, calcium sulfate,
dihydrate, terra alba, magnesium stearate or stearic acid, talc,
pectin, acacia, agar or gelatin. The carrier may also include a
sustained release material such as glyceryl monostearate or
glyceryl distearate, alone or with a wax.
The pharmaceutical compositions according to the invention may be
delivered in a therapeutically effective amount. The precise
therapeutically effective amount is that amount of the composition
that will yield the most effective results in terms of efficacy of
treatment in a given subject. This amount will vary depending upon
a variety of factors, including but not limited to the
characteristics of the therapeutic compound (including activity,
pharmacokinetics, pharmacodynamics, and bioavailability), the
physiological condition of the subject (including age, sex, disease
type and stage, general physical condition, responsiveness to a
given dosage, and type of medication), the nature of the
pharmaceutically acceptable carrier or carriers in the formulation,
and the route of administration. One skilled in the clinical and
pharmacological arts will be able to determine a therapeutically
effective amount through routine experimentation, for instance, by
monitoring a subject's response to administration of a compound and
adjusting the dosage accordingly. For additional guidance, see
Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th
edition, Williams & Wilkins PA, USA) (2000). In one embodiment,
the pharmaceutical composition comprises Pgp.sup.+ or Pgp.sup.-
cells at 1.times.10.sup.5 to 1.times.10.sup.8 cells/ml (or any
value there between which is expressly contemplated herein). The
cells may be introduced directly into the peripheral blood or
deposited within other locations throughout the body, e.g., a
desired tissue, or on microcarrier beads in the peritoneum. For
example, 10.sup.2 to 10.sup.11 cells can be transplanted in a
single procedure, and additional transplants can be performed as
required.
Cells can be engineered using any of a variety of vectors
including, but not limited to, integrating viral vectors, e.g.,
retroviral vectors or lentiviral vectors or adeno-associated viral
vectors; or non-integrating replicating vectors, e.g., papilloma
virus vectors, SV40 vectors, adenoviral vectors; or
replication-defective viral vectors. Where transient expression is
desired, non-integrating vectors and replication defective vectors
may be used, since either inducible or constitutive promoters can
be used in these systems to control expression of the gene of
interest. Where the vector is a non-integrating vector, such
vectors can be lost from cells by dilution, as desired. An example
of a non-integrating vector includes Epstein-Barr virus (EBV)
vector. Alternatively, integrating vectors can be used to obtain
transient expression, provided the gene of interest is controlled
by an inducible promoter. Other methods of introducing DNA into
cells include the use of liposomes, lipofection, electroporation, a
particle gun, or by direct DNA injection. Alternatively, cells can
be engineered using transfection of in vitro transcribed mRNA.
Conventional recombinant DNA techniques can be used in the methods
of the disclosure. For example, conventional recombinant DNA
techniques are used to introduce a desired polynucleotide into
cells (e.g., polynucleotides encoding a CAR). The precise method
used to introduce a polynucleotide is not critical to the
disclosure. For example, physical methods for the introduction of
polynucleotides into cells include microinjection and
electroporation or viral gene therapy. Chemical methods such as
coprecipitation with calcium phosphate and incorporation of
polynucleotides into liposomes are also standard methods of
introducing polynucleotides into mammalian cells. For example, DNA
or RNA can be introduced using standard vectors, such as those
derived from murine and avian retroviruses (see, e.g., Gluzman et
al., 1988, Viral Vectors, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.) or lentiviral vector systems. Standard
recombinant molecular biology methods are well known in the art
(see, e.g., Ausubel et al., 1989, Current Protocols in Molecular
Biology, John Wiley & Sons, New York), and viral vectors for
gene therapy have been developed and successfully used clinically
(Rosenberg, et al., 1990, N. Engl. J. Med, 323:370). Other methods,
such as naked polynucleotide uptake from a matrix coated with DNA
are also encompassed by the disclosure (see, for example, U.S. Pat.
No. 5,962,427, which is incorporated herein by reference).
Any promoter may be used to drive the expression of the inserted
gene. For example, viral promoters include but are not limited to
the CMV promoter/enhancer, SV40, papillomavirus, Epstein-Barr
virus, elastin gene promoter and beta-globin. If transient
expression is desired, constitutive promoters are used in a
non-integrating and/or replication-defective vector. Alternatively,
inducible promoters could be used to drive the expression of the
inserted gene when necessary. Inducible promoters can be built into
integrating and/or replicating vectors. For example, inducible
promoters include, but are not limited to, metallothionien and heat
shock protein.
The lymphocytes or progenitor lymphocytes of the disclosure can be
isolated from a sample obtained from a mammalian subject. The
subject can be any mammal (e.g., bovine, ovine, porcine, canine,
feline, equine, primate), including a human. The sample of cells
may be obtained from any of a number of different sources
including, for example, bone marrow, fetal tissue (e.g., fetal
liver tissue), peripheral blood, umbilical cord blood, healthy or
diseased tissue (e.g., tumor infiltrating lymphocytes) and the
like.
Although the disclosure has exemplified in various embodiments
Pgp.sup.+ and Pgp.sup.- T cells, the methods and compositions of
the disclosure are applicable to NK cells, Pgp.sup.+ hematopietic
stem cells (e.g., CD34.sup.+/Pgp.sup.+ hematopoietic stem cells)
and the like. Thus, in any of the various embodiments describe
herein and throughout the specification the term "T cell" can be
replaced with "NK cell" or "CD34.sup.+ cell" etc. One of skill in
the art will recognize that CD34.sup.+ cells are stem cells and
thus have additional methods and compositions applications that
extend beyond T cell. For example, CD34.sup.+ cells can be
isolated/purified using metabolic starvation, hyperthermia and/or
chemotoxic compounds and used for allogeneic or autologous stem
cell transplantation with or without genetic modifications to the
cells.
In another embodiment, the disclosure provides methods of
establishing and/or maintaining populations of cells, or the
progeny thereof, as well as mixed populations comprising various
cells types as well as subpopulations (e.g., Pgp.sup.+ and/or
Pgp.sup.-). Once a culture of cells or a mixed culture of cells
and/or progenitor cell (stem-like cells) is established, the
population of cells is maintained and/or mitotically expanded in
vitro by passage to fresh medium as cell density dictates under
conditions conducive to cell proliferation and maintenance.
Once cells or desired sub-population of cells of the disclosure
have been established in culture, as described above, they may be
maintained or stored in cell "banks" comprising either continuous
in vitro cultures of cells requiring regular transfer or cells
which have been cryopreserved.
Cryopreservation of cells of the disclosure may be carried out
according to known methods, such as those described in Doyle et
al., (eds.), 1995, Cell & Tissue Culture: Laboratory
Procedures, John Wiley & Sons, Chichester. For example, but not
by way of limitation, cells may be suspended in a "freeze medium"
such as, for example, culture medium further comprising 15-20%
fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO), with or
without 5-10% glycerol, at a density, for example, of about
4-10.times.10.sup.6 cells/ml. The cells are dispensed into glass or
plastic vials which are then sealed and transferred to a freezing
chamber of a programmable or passive freezer. The optimal rate of
freezing may be determined empirically. For example, a freezing
program that gives a change in temperature of -1.degree. C./min
through the heat of fusion may be used. Once vials containing the
cells have reached -80.degree. C., they are transferred to a liquid
nitrogen storage area. Cryopreserved cells can be stored for a
period of years, though they should be checked at least every 5
years for maintenance of viability.
The cryopreserved cells of the disclosure constitute a bank of
cells, portions of which can be withdrawn by thawing and then used
to produce a cell culture as needed. Thawing should generally be
carried out rapidly, for example, by transferring a vial from
liquid nitrogen to a 37.degree. C. water bath. The thawed contents
of the vial should be immediately transferred under sterile
conditions to a culture vessel containing an appropriate medium. It
is advisable that the cells in the culture medium be adjusted to an
initial density of about 1-3.times.10.sup.5 cells/ml. Once in
culture, the cells may be examined daily, for example, with an
inverted microscope to detect cell proliferation, and subcultured,
if appropriate, as soon as they reach an appropriate density.
In another embodiment, the disclosure provides cell lines of
Pgp.sup.+ cells. As used herein a "cell line" means a culture of
cells of the disclosure, or progeny cells thereof, that can be
reproduced for an extended period of time, preferably indefinitely,
and which term includes, for example, cells that are cultured,
cryopreserved and re-cultured following cryopreservation. As used
herein a "culture" means a population of cells grown in a medium
and optionally passaged accordingly.
The following examples are intended to illustrate particular
embodiments and not to limit the scope of the disclosure.
EXAMPLES
Example 1
Tetramethylrhodamine methyl ester (TMRM) was purchased from
Thermofisher and dissolved in DMSO. DiOC2(3) was purchased from
Life Technologies and dissolved in methanol to produce 1 mg/ml
stock solution. TH9402 was synthesized by the Weill Cornell
Medicine Milstein Chemistry Core Facility and dissolved in DMSO to
generate 100 mM stock. Unconjugated and FITC-conjugated monoclonal
antibody (MoAb) UIC2 (IgG2a) against an extracytoplasmic domain of
human Pgp was obtained from Santa Cruz Biotechnology. Fluorescein
isothiocyanate--(FITC)-conjugated goat antimouse IgG was from
Southern Biotechnology. Buffy coat cells were obtained from healthy
de-identified adult donors from the Blood Bank at Children Hospital
of Los Angeles and used to isolate peripheral blood mononuclear
cells (PBMC) by Ficoll-Hypaque gradient centrifugation. PBMC were
either used as such or used to isolate T cells using CD3 magnetic
microbeads (Miltenyi Biotech) and following the manufacturer's
instructions. PBMC or isolated T cells were re-suspended in XVIVO
medium (Lonza) supplanted with 10 ng/ml soluble anti-CD3, 10 ng/ml
soluble anti-CD28 and 100 IU recombinant human-IL2 unless indicated
otherwise. Cells were cultured at 37.degree. C., in a 5% CO.sub.2
humidified incubator, unless indicated otherwise.
Approximately 15 million PBMC cells were stained with TMRM (20 nM
final concentration) in XVIVO medium for 30 min at 37.degree. C. in
the absence or presence of Reserpine (50 .mu.l of 1 mM stock added
to 5 ml of medium with cells to give final concentration of 10
.mu.M) or cyclosporine (10 .mu.l of 1 mM stock added to 5 ml of
medium with cells; final concentration=2 .mu.M). All subsequent
steps were done with cells maintained at 4.degree. C. in dark.
Cells were centrifuged and cell pellets were blocked with 200 .mu.l
of human AB serum for 1 hr at 4.degree. C. Cells were washed with
ice cold PBS containing 1% FCS, and divided into 3 tubes. Cells in
each tube were centrifuged and cell pellets incubated for
approximately 2 h in dark on ice with 1) 10 .mu.l of
FITC-conjugated UIC2 (MDR-1) antibody (Santa Cruz Biotechnology;
SC-73354), 2) 1 .mu.g of unconjugated UIC2 antibody (200 .mu.g/ml;
Santa Cruz Biotechnology; SC-73354) or 3) 1 .mu.g of unconjugated
isotype control (mouse IgG2a) antibody (eBioscience; Ref
14-4732-85). Cells labeled with unconjugated UIC2 and isotype
control antibody were washed twice with PBS containing 1% FCS,
centrifuged and cell pellet labeled with 5 .mu.l (2.5 .mu.g) of
FITC-conjugated Goat F(ab)2 anti-mouse IgG (H+L) human adsorbed
antibody (Southern Biotechnology; Cat#1032-02). Cells were washed
twice with PBS containing 1% FCS and analyzed using BD verse flow
cytometer. Cell fluorescence was analyzed in lymphoid gate based on
forward and side scatter, thereby focusing the analysis on
peripheral blood lymphocytes (PBL).
When stained at 37.degree. C. in the absence of P-glycoprotein
inhibitors reserpine and cyclosporine, 50% to 70% of lymphocytes
were found to be TMRM-dull. In contrast, only a minor population of
lymphocytes were TMRM-dull when stained in the presence of
reserpine and cyclosporine, suggesting that a reserpine- and
cyclosporine-sensitive efflux pump is responsible for the TMRM-dull
phenotype of the vast majority of lymphocytes.
In addition to P-glycoprotein, a number of other ABC transporters
have been described that are capable of pumping out lipophilic
drugs, including dyes. To determine if the TMRM-dull phenotype of
lymphocytes correlates with P-glycoprotein expression, double
staining with TMRM and FITC-conjugated UIC2 antibody was used.
However, no correlation between TMRM staining and Pgp expression,
as determined by staining with FITC-UIC2 was observed. The lack of
correlation between TMRM staining and FITC-UIC2 could be due to low
level Pgp expression in lymphocytes which was not detected by
FITC-conjugated UIC2 antibody. To increase the sensitivity of Pgp
detection, the above experiment was repeated by performing indirect
immunofluorescence labeling with UIC2 antibody. The use of indirect
labeling results in increased sensitivity as each molecule of UIC2
antibody can be potentially bound by many molecules of FITC-labeled
secondary antibody. Additionally, to further improve the
sensitivity of Pgp expression, the amount of secondary antibody
used was increased and the incubation volume was reduced, thereby
resulting in high concentration of the secondary antibody. The
incubation time was also increased to 2 h. Finally, to reduce
non-specific binding of the secondary antibody, F(ab).sub.2
fragment (instead of whole antibody) that had been adsorbed against
human serum proteins was used. Using this optimized staining
protocol, robust expression of Pgp was observed in lymphocytes.
More importantly, there was a strong inverse correlation between
the levels of P-gp expression, as measured by UIC2 staining, and
the retention of TMRM in the lymphocytes, indicating that the dye
efflux was directly correlated with P-gp expression (FIG. 1F).
Fluorescence activated cell sorting (FACS) analysis was also
conducted on cells that had been stained with TMRM in the presence
of Pgp inhibitors (10 .mu.M Resperine and 2 .mu.M cyclosporine) and
then stained with UIC2 followed by FITC conjugated Goat antimouse
IgG. A vast majority of Pgp-expressing cells stained brightly with
TMRM under these conditions (FIG. 1H, 1I, 1K, 1L). These results
demonstrate that (i) Pgp expression has a major influence on TMRM
staining of PBL; and (ii) metabolic state, as measured by
mitochondrial membrane potential, is not the only determinant of
TMRM staining.
Example 2. Using Immunofluorescence Staining to Enrich for Pgp
Expressing Cells
Peripheral blood mononuclear cells (10 million cells) were stained
as described in the previous example except three monoclonal
antibodies against Pgp, including UIC2, MRK16, and 4E3 are used as
primary antibodies (each at a concentration of 0.5 .mu.g/million
cells) to increase the sensitivity of the assay. Following
extensive staining, cells were stained with FITC conjugated 5 .mu.l
(2.5 .mu.g) of FITC-conjugated Goat F(ab)2 anti-mouse IgG (H+L)
human adsorbed antibody (Southern Biotechnology; Cat#1032-02).
Cells were washed and free antibody binding sites were blocked by
addition of mouse IgG. After 2 washes, cells were labeled with
PE-conjugated human CD8 antibody and APC-conjugated CD4 antibody
for 1 h at 4.degree. C. Cells were analyzed by Flow cytometry and
sorted into different fractions (e.g Pgp.sup.+,
Pgp.sup.+/CD8.sup.+, Pgp.sup.+/CD4.sup.+, Pgp.sup.+/CD8.sup.-,
Pgp/CD4.sup.-, Pgp.sup.-/CD8.sup.-, Pgp.sup.-/CD4.sup.-).
Example 3. Using MACS (Magnetic Activated Cell Sorting) to Enrich
for Pgp Expressing Cells
Protocol 1.
The following protocol was used to enrich Pgp expressing cells by
MACS on staining with Pgp specific monoclonal antibodies and Goat
anti-mouse IgG2a+IgG2b magnetic beads (Miltenyi). The blood samples
to isolate peripheral blood mononuclear cells (PBMCs) were obtained
from healthy de-identified adult donors. PBMC were isolated from
buffy coats by Ficoll-Hypaque gradient centrifugation.
Approximately 50 million PBMCs growing in XVIVO medium supplemented
with hIL2 were stained with TMRM (20 nM) for 30 min at 37.degree.
C. Cells were centrifuged and cell pellet blocked with 500 .mu.l of
human serum for 1 hour at 4.degree. C. TMRM-stained 50 million
cells were separated into 3 tubes as follows: Tube 1: Stained with
Pgp-UIC2 (unconjugated) antibody (1 .mu.g/1 million cells) from
Santa Cruz Biotech; Tube 2: stained with an Pgp-4E3 antibody (1
.mu.g/1 million cells) from Abcam; Tube 3: stained with both UIC2
and 4E3 antibodies each at 1 .mu.g/1 million cells. Cells were
incubated with the above antibodies for 2 hours at 4.degree. C. 2
ml of MACS buffer was added to each tube, cells were centrifuged at
300.times.g for 10 min at 4.degree. C. and resuspended in 80 .mu.l
MACS buffer/tube. 20 .mu.l of anti-Mouse IgG2a+b microbeads
(Miltenyi: 130-047-201) were added to each tube and cells incubated
at 4.degree. C. for 30 min. 2 ml of MACS buffer was added to each
tube, cells centrifuged and resuspended into 500 .mu.l MACS buffer.
Cells were loaded cells on pre-washed MS columns (Miltenyi).
Flow-through fraction was collected as negative cell fraction and
column-bound cells were eluted as MDR1.sup.+ (Pgp.sup.+) cells
following manufacturer's instructions. Aliquots of the positive and
negative cells were analyzed by flow cytometer.
The results showed that there was enrichment for TMRM-dull cells
(representing Pgp expressing cells) in the cells isolated based on
UIC2 and 4E3 staining alone. More importantly, there was greater
enrichment for TMRM-dull cells among the cells isolated based on
simultaneous staining with both UIC2 and 4E3. The above results
demonstrated that TMRM-dull cells can be purified based on staining
with Pgp-specific antibodies that bind to the extracellular domain
of Pgp. Furthermore, combination of Pgp specific antibodies,
particularly those that bind to different epitopes of Pgp, can be
used to obtain higher yield and greater purity. In addition to UIC2
and 4E3, a number of other Pgp antibodies are commercially
available (e.g. MRK16, REA495) and can be used either alone or in
combination to purify Pgp expressing (TMRM-dull) cells for the
purpose of cellular therapies. Polyclonal antibodies against the
extracellular domain of Pgp can be also used for the purpose of
this disclosure. A rabbit polyclonal against human MDR1 protein is
available from Bioss Inc. Finally, other Pgp binding moieties, such
as scFv, single domain antibodies, F(ab)2 fragments, affibodies,
nanobodies etc., can be used for the purpose of this invention.
These antibodies (monoclonal and polyclonal) and antibody like
moieties, singly or in combination, can be also used to deplete
Pgp-expressing cells from a starting cell population for the
purpose of cell therapies where it is desirable to deplete
Pgp-expressing cells, such as to reduce Graft vs host disease in
patients undergoing allogeneic stem cell transplant.
Example 4. Purification of Pgp Expressing Cells from Peripheral
Blood Mononuclear Cells Using MACS
Protocol 2.
To enhance the purity and yield of Pgp expressing cells, the
procedure in the preceding example is repeated using a cocktail of
monoclonal antibodies against Pgp including UIC2, 4E3, REA495 and
MRK16. Each antibody is used at 0.5 .mu.g/million cells. Staining
with primary antibodies is carried out at 4.degree. C. for 1 h and
after extensive washes the cells are incubated with 50 .mu.l of
anti-Mouse IgG beads/million cells for 2-4 hr with intermittent
shaking. Positive and negative fractions are isolated as described
in the previous section. The modified procedure is shown to result
in greater yield and purity of Pgp-expressing cells.
Example 5. Purification of Pgp Expressing Cells from Peripheral
Blood Mononuclear Cells Using MACS
Protocol 3. Biotinylated REA495 antibody against Pgp and
streptavidin microbeads were purchased from Miltenyi Biotech. PBMC
or T cells is labeled with Biotinylated REA495 at 4.degree. C. for
1 h following manufacturer's recommendation. After extensive washes
with labeling buffer (Miltenyi Biotech), the cells are resuspended
in 90 .mu.l of labeling buffer per 10.sup.7 cells. Then, 40 .mu.l
of streptavidin microbeads are added to the cells. Cells are mixed
and refrigerated at 4-8.degree. C. for 1-2 hr with intermittent
shaking. Cells are washed with 1-2 ml of buffer and centrifuged at
300 g for 10 min at 4-8.degree. C. Cells are resuspended in 500
.mu.l of separation buffer and used for magnetic separation
following the recommendations of the manufacturer. Positive and
negative fractions are isolated as described in the previous
section.
Example 6. Purification of Pgp Expressing Cells from T Cells Using
MACS
Protocol 4.
In the preceding examples, Pgp expressing cells were isolated from
Peripheral blood mononuclear cells (PBMC) that were obtained from
Ficoll-Hypaque separation. To purify Pgp expressing T cells, PBMC
are enriched for T cells using a Pan T cell Isolation kit (Catalog
#130-096-535) available from Miltenyi and following the
manufacturer's recommendations. This kit uses a cocktail of
antibodies against markers that are not present on T cells to
deplete cells belonging to other lineages. Similar kits are
available from other sources as well. The T cell enriched fraction
is then positively selected for Pgp expressing cells using the
protocol 1, 2 or protocol 3 described above.
Example 7. Purification of Pgp Expressing Cells from PBMC Using
Photodynamic Cell Therapy with TH9402
T cells were isolated using PAN T-cell isolation kit (Miltenyi cat
no. 130-096-535) from buffy coat preparation following
Ficoll-Hypaque separation and RBC lysis as described above. 12
million T cells were resuspended at 1 million cells/ml in XVIVO T
cell medium (Lonza) supplemented with 5 ng/ml IL7. Half (6 million)
of the T cells were left untreated while the remaining half were
treated with 10 .mu.M of TH9402 compound. Cells were incubated at
37.degree. C. in a water bath in dark for 40 min, washed with
T-cell medium and then resuspended in TH9402-free T-cell medium.
Cells were allowed to efflux TH9402 at 37.degree. C. in dark in 10
ml of T cell medium for 2 h. Cells were centrifuged and
re-suspended in fresh medium. Each sample was then plated in 2
wells of two different 6-well plates. One plate was left unexposed
to light and the second plate was exposed for 1 h to light
(1000.times.10 Lux units) from an LED lamp. Alternatively, light
treatment can be achieved by exposure to a fluorescent
light-scanning device (PDCT-Xerox Series 4, Theratechnologies)
delivering 5 J/cm.sup.2 at wavelength of 514 nm. After light
exposure, cells were incubated in the XVIVO T cell medium with 5
ng/ml IL7 at 37.degree. C. for 2 days in a 5% CO.sub.2 incubator.
After 2 days, an aliquot of the cells were stained at 4.degree. C.
for 40 min with DiOC2(3) (60 ng/ml) in 10 ml RPMI with 10% FBS
medium. DiOC2(3) is a known substrate of Pgp. Cells were
centrifuged, washed, resuspended in 10 ml of dye-free RPMI-10% FBS
medium to efflux dye at 37.degree. C. for 90 min. After Dye efflux,
the cells were stained with Propidium iodide (PI) and examined by
flow cytometry. The percentage of cells in the lymphoid gate in the
various treatment groups are shown in the following table. The
results show that even in the group which was untreated with TH904,
there is significant enrichment for P-glycoprotein expressing
lymphoid cells that stain dull with DiOC2(3) upon exposure to light
(12% vs 62%), suggesting that light exposure, by itself, can lead
to enrichment of Pgp-expressing T lymphocytes. Furthermore, in the
group that was treated with the TH9402 compound and then exposed to
light, there was further enrichment for Pgp-expressing T cells
(from 10% to 80%).
Essentially similar results were obtained when the experiment was
repeated with PBMC rather than purified T cells.
TABLE-US-00001 % live % Pgp.sup.+ cells (DiOC2(3)- Sample cells
(P1) dull) T-Untreated (UT)- 75 12 unexposed T-TH9402-treated- 78
10 unexposed T-UT-1 h exposure to 21 62 light T-TH9402-1 h exposure
to 10 80 light
After 6 days, 250 .mu.l cell aliquot from T cells were stained with
DiOC2(3), allowed to efflux the dye and then stained with
CD62L-APC, a marker present on human memory stem T cells
(T.sub.SCM) with stem like properties.
TABLE-US-00002 % Pgp.sup.+ (DiOC2(3)- % Pgp.sup.+ dull cells
(DiOC2(3)- (based on dye dull) efflux) (gated CD62L + cells % live
on live (gated on Sample cells (P1) cells) live cells)
T-UT-unexposed 77 5 3 T-TH-unexposed 75 4 2 T-UT-1 h exposure to 24
6 3 light T-TH9402-1 h exposure to 14 70 69 light
After 9 days, 1 ml cell aliquot from T cells were stained with
DiOC2(3), allowed to efflux the dye and then stained with
CD62L-APC, a marker present on human memory stem T cells
(T.sub.SCM) with stem like properties. Cells were then analyzed by
flow cytometry. The % of cells in the different fractions are shown
in FIG. 2 and the following table which show there is a significant
decline in cell viability following exposure to light in both
TH9402-treated and untreated cells. However, among the live cells
that had been treated with TH9402 and then exposed to light, there
is a significant enrichment (from 45% to 79%) for cells that
express Pgp (i.e. that are DiOC2(3)-dull) as compared to cells that
had not been treated with TH9402 but were then exposed to light.
Furthermore, among the live cells that had been treated with TH9402
and then exposed to light, there is a significant enrichment (from
21% to 67%) for cells that express both Pgp (i.e. that are
DiOC2(3)-dull) and CD62L as compared to cells that had not been
treated with TH9402 but were then exposed to light.
TABLE-US-00003 % Pgp.sup.+ (DiOC2(3)- % Pgp.sup.+ % live dull cells
(DiOC2(3)- cells (Based on dull), (Propidium dye efflux) CD62L +
cells iodide (gated on (gated on live Sample negative) live cells)
cells) T-UT-unexposed 75 46 19 T-TH9402-unexposed 79 45 21 T-UT-1 h
exposure to 38 50 49 light T-TH9402-1 h exposure 27 79 67 to
light
Example 8. Sorting of Pgp.sup.+ Cells, Expression of CAR in Sorted
Cells
T Cells were isolated form donor blood. PBMC were isolated after
RBC lysis and Ficoll gradient. 130 million T cells were isolated
from 400 million PBMCs using T-PAN isolation kit, cat no.
130-096-535 from Miltenyi. T cells were c.mu.ltured in XVIVO medium
with IL2 100 IU/ml. CD3 or CD28 antibodies were not added.
100 million T cells were washed with PBS +1% hAB serum at 4.degree.
C. and blocked with 500 .mu.l hAB serum at 4.degree. C. for 1 h
followed by washing (2 times). Mixture of two primary antibodies to
stain (Pgp.sup.+) MDR+ cells were used: MDR (UIC2) sc-73354 0.5 g
per million cells and P-glycoprotein antibody from Abcam, Ab 10333
0.2 .mu.g per million cells, were used and incubated at 4.degree.
C. for 1 h followed by washing twice at 4.degree. C.
Secondary antibody Goat F(ab').sub.2-a-mouse-IgG(H+L) human
ads-FITC cat no 1032-02, Southern Biotech is prepared as a stock
0.5 mg/ml. For 100 million T cells, 250 .mu.l of secondary antibody
was added to cell pellets re-suspended in residual wash buffer, no
extra buffer added. The pellets were stained for 2 hours, with
intermittent shaking at 4.degree. C., followed with two washings.
The cells were re-suspended in PBS with 1 .mu.g/ml Propidium Iodide
(PI) to exclude dead cells. Cells were checked on flow for FITC
staining before sorting.
Cells were sorted on a MoFlo machine. Dead, PI+, cells were
excluded and FITC.sup.+ and FITC.sup.- cells collected. 9 million
FITC.sup.+ cells and 7.5 million FITC.sup.- cells were collected,
cultured in 6 well plates with complete T cells medium supplemented
with IL2 (100 IU/ml), and CD3 (30 ng/ml) and CD28 (30 ng/ml)
antibodies.
Pgp.sup.+ and Pgp.sup.- cells were infected with FMC63-BBz-A13 CAR
virus, by infection three times on three days using Polybrene at 18
.mu.l per well. Spinfection was performed at 2800 rpm, 32.degree.
C. for 90 min, and the medium was replaced with fresh medium after
6 h of infection.
Cells were expanded without selection with puromycin in IL2, CD3,
and CD28 containing T cell medium. MDR.sup.+ sorted cells formed
bigger clumps as compared to MDR.sup.- cells. Thus showing that
Pgp.sup.+ cells proliferate more compared to Pgp.sup.- cells
TABLE-US-00004 Total cell Total cell Total cell Sample count-4 days
count-6 days count-8 days Pgp.sup.+ sorted-FMC63- 7 million 12.2
million 12 million BBz-A13 Pgp.sup.- sorted FMC63- 1.26 million
0.42 million 0 million BBz-A13 cells
Example 9. Purification of Pgp Expressing Cells by Chemotherapeutic
Selection Criteria
T cells were isolated using CD3 microbeads. Cells were resuspended
in T cell culture medium and treated under the following
conditions.
1. High dose Vincristine Treatment: Cells were treated with
Vincristine (Signma) at doses of 100, 250, 500, 750, 1000 ng/ml for
24 h. The following day, the medium was changed and cells cultured
in Vincristine-free medium.
2. Intermediate Dose Vincristine Treatment: Cells were treated with
vincristine at intermediate doses of 5, 10, 20, 30, 50 ng/ml with
continuous exposure of the drug thereafter.
3. Low dose Vincristine Treatment: Cells were treated with
vincristine at low doses 0.5, 1, 2, 2.5, 3 ng/ml with continuous
exposure to the drug thereafter.
4. Cells were treated with Akt inhibitor vIII (Cat#124018,
Calbiochem), at 1 .mu.M final conc.
5. Cells were treated with Etoposide at dose 100, 500, 1000 nM
6. Cells were treated with Adriamycin (doxorubicin) at 0.1, 0.5, 1
.mu.g/ml
T cells treated with intermediate and high dose of vincristine were
checked for Pgp.sup.+ enrichment by DiOC2(3) efflux assay and cell
death by Propidium iodide staining after 2 days of treatment. Cells
were analyzed by flow cytometry. The results as shown in the table
below demonstrate a modest enrichment of Pgp.sup.+ cells with
higher dose vincristine treatments after 2 days.
TABLE-US-00005 % Pgp.sup.+ (DiOC2(3)- Samples: 2 days post- % Cell
Death (PI+ dull) (out of live treatment cells) cells) Untreated 14
8 Vincristine-5 ng/ml 13 8 Vincristine-10 ng/ml 15 9 Vincristine-20
ng/ml 15 9 Vincristine-30 ng/ml 20 11 Vincristine-50 ng/ml 15 10
Vincristine-100 ng/ml 28 11 Vincristine-250 ng/ml 31 11
Vincristine-500 ng/ml 27 13 Vincristine-750 ng/ml 30 14
Vincristine-1 .mu.g/ml 31 14
T cells treated with low, intermediate and high dose of vincristine
were checked for Pgp.sup.+ enrichment by DiOC2(3) efflux assay and
cell death by Propidium iodide staining after 2 days of treatment
and after 7 days of treatment. The results are shown in the
following table and demonstrate that after 7 days of vincristine
treatment, there was a significant enrichment of Pgp.sup.+ cells
with 1 .mu.g/ml Vincristine from 5% in untreated group to 18% in
the 1 .mu.g/ml-treated group.
TABLE-US-00006 % Pgp+ (DiOC2(3)- Samples: 7 days post- % Cell Death
(PI+ dull) (gated on live treatment cells) cells) UT 9 5
Vincristine-0.5 ng/ml 10 7 Vincristine-1 ng/ml 12 7 Vincristine-2
ng/ml 8 7 Vincristine-2.5 ng/ml 10 7 Vincristine-3 ng/ml 14 9
Vincristine-5 ng/ml 9 8 Vincristine-10 ng/ml 9 10 Vincristine-20
ng/ml 9 10 Vincristine-50 ng/ml 14 9 Vincristine-100 ng/ml 16 13
Vincristine-250 ng/ml 25 11 Vincristine-500 ng/ml 26 9
Vincristine-750 ng/ml 29 10 Vincristine-1 .mu.g/ml 23 18
T cells treated with drugs other than vincristine were checked for
Pgp.sup.+ enrichment after 8 days of treatment using the assay
described above. Treatment with adrimycin (doxorubicin) at 1
.mu.g/ml showed significant enrichment of Pgp.sup.+ or
DiOC2(3)-dull cells.
TABLE-US-00007 Sample (8 days post- % Cell Death (PI+ % pgp+ (out
of live treatment) cells) cells) UT 3 16 Rapamycin-100 ng/ml 11 15
RAD1001-0.2 ng/ml 7 15 RAD1001-1 ng/ml 5 15 Adriamycin-0.1 .mu.g/ml
5 16 Adriamycin-0.5 .mu.g/ml 7 16 Adriamycin-1 .mu.g/ml 12 27
Etoposide-0.1 uM 5 15 Etoposide-0.5 unM 4 16 Etoposide-1 uM 5 15
Akt-inhibitor-1 uM 4 19 Verapamil-1 uM 4 17 Verapamil-5 uM 3 17
Verapamil-10 uM 3 17
Example 10. Temperature Selection of Pgp-Expression Cells
T cells were isolated as described above. Three different water
baths were set up at 42, 43 and 44.degree. C. Four 6-well plates
labelled as 37, 42, 43 and 44 containing 1 ml of T-cell medium were
kept at 37.degree. C. incubator to pre-warm the media. T cells (1
million cells/ml) in 1 ml of T-cell medium with IL7 (5 ng/ml) were
kept at 37.degree. C. or in the above water baths for the indicated
time intervals as follows:
1. 37.degree. C.
2. 42.degree. C. for 1 h
3. 42.degree. C. for 2 h
4. 42.degree. C. for 3 h
5. 42.degree. C. for 4 h
6. 42.degree. C. for 5 h
7. 42.degree. C. for 6 h
8. 43.degree. C. for 1 h
9. 43.degree. C. for 2 h
10. 43.degree. C. for 3 h
11. 43.degree. C. for 4 h
12. 43.degree. C. for 5 h
13. 43.degree. C. for 6 h
14. 44.degree. C. for 1 h
15. 44.degree. C. for 2 h
16. 44.degree. C. for 3 h
17. 44.degree. C. for 4 h
18. 44.degree. C. for 5 h
19. 44.degree. C. for 6 h
After each time point, cells were added to separate wells of 6 well
plates containing the pre-warmed medium and kept at 37.degree. C.
for rest of the experiment.
After 5 days the percentage of Pgp.sup.+ cells were checked by
DiOC2(3) efflux to check if exposure of T cells to high temperature
for short time point can enrich Pgp.sup.+ cell population. The
results are shown in the following Table and demonstrate
significant enrichment of Pgp.sup.+ (DiOC2(3)-dull) cells following
exposure to elevated temperatures. For example, DiOC2(3)-dull cells
showed enrichment from 76% to 98% when exposed to 43.degree. C. for
2 hours as compared to cells kept at 37.degree. C.
TABLE-US-00008 Pgp+ (DIOC2- effluxing cells (gated on live Sample
Live cells (%) cells) T-37.degree. C. 65 76 T-42.degree. C.-1 h 62
77 T-42.degree. C.-2 h 67 96 T-42.degree. C.-3 h 50 93 T-43.degree.
C.-1 h 60 93 T-43.degree. C.-2 h 20 98 T-43.degree. C.-3 h 4 95
Peripheral blood stem cell cells were obtained from a patient
undergoing stem cell transplantation. Cells underwent RBC lysis to
get rid of red cells and Ficoll-Hypaque separation to enrich for
mononuclear cells. Approximately, 10 million cells were recovered
and approximately 7 million cells were used for hyperthermia
experiment. Cells were resuspended in 15 ml Falcon tubes at
approximately 1 million cells/ml and were kept at 37.degree. C. or
in a water-bath at 43.degree. C. for the indicated time intervals
as follows:
37.degree. C.
43.degree. C. for 0.5 h
43.degree. C. for 1 h
43.degree. C. for 1.5 h
43.degree. C. for 2 h
43.degree. C. for 2.5 h
43.degree. C. for 3 h
Following exposure cells were transferred to a 6 well plate at
incubated at 37.degree. C. in Stem-cell medium XVIVO-10
supplemented with SCF, TPO, FLT3, IL3, IL6 (all at 50 ng/ml) in a
humidified 5% CO.sub.2 incubated for 72 h. 100 .mu.l aliquots were
then stained with 1 .mu.g/ml Propidium iodide to check cell
death.
TABLE-US-00009 % lymphocytes (P1 Sample population) % PI+ve dead
cells T-37.degree. C. 18 12 T-43.degree. C.-0.5 h 16 13
T-43.degree. C.-1 h 8 23 T-43.degree. C.-1.5 h 6 31 T-43.degree.
C.-2 h 7 29 T-43.degree. C.-2.5 h 5 39 T-43.degree. C.-3 h 5 36
After 96 hours, cells were stained with DiOC2(3) (60 ng/ml in 5 ml
of RPMI 10% FBS medium at 4.degree. C. for 40 min). The cells were
washed with medium, dye-efflux in 10 ml RPMi 10% medium at
37.degree. C. for 90 min, washed twice with PBS 1% FBS, and stained
with 1.5 .mu.l/sample/100 .mu.l of CD34-APCefluor 780 (ebiosciences
cat#470349-42) at 4.degree. C. for 1 h. The cells were washed and
analyzed by Flow Cytometry. APC-efluor-780 was detected in APC-Cy7
channel in BD Facsverse. The results demonstrate significant
enrichment of Pgp.sup.+ cells from 48% to 76-80% following exposure
to 43.degree. C. for different time intervals. In addition there is
enrichment of Pgp.sup.+/CD34.sup.+ stem cells from 1% to 2% in
cells exposed to 43.degree. C. for 3 h as compared to cells
cultured at 37.degree. C.
TABLE-US-00010 % Pgp.sup.+ (DiOC2- Sample effluxing cells) %
Pgp.sup.+CD34.sup.+ T-37.degree. C. 48 1 T-43.degree. C.-0.5 h 76
0.3 T-43.degree. C.-1 h 70 1 T-43.degree. C.-1.5 h 76 0.5
T-43.degree. C.-2 h 76 1 T-43.degree. C.-2.5 h 76 0.5 T-43.degree.
C.-3 h 80 2
Example 11. Use of Pgp Enriched Cells for Adoptive Cellular
Therapy
The blood samples to isolate peripheral blood mononuclear cells
(PBMCs) are obtained from healthy de-identified adult donors. PBMC
are isolated from buffy coats by Ficoll-Hypaque gradient
centrifugation. Pgp expressing cells are purified from PBMC by any
one or more of the methods described in the previous examples,
including Flow sorting, MACS and photodynamic cell therapy with
TH9402, selection with vincristine and hyperthermia. Pgp.sup.+ T
cells, Pgp.sup.- T cells and unpurified T cells are re-suspended in
XVIVO medium (Lonza) supplanted with 10 ng/ml soluble anti-CD3, 10
ng/ml soluble anti-CD28 and 100 IU recombinant human-IL2. Cells are
engineered to express FMC63(vL-vH)-Myc-BBz-PAC Chimeric Antigen
Receptor (CAR) targeting human CD19 by infection with
pLENTI-EF1a-FMC63(vL-vH)-Myc-BBz-T2A-Pac-A13 lentiviral vector. NSG
mice (Jackson Lab) are sub-lethally irradiated at a dose of 175
cGy. 24 hours post irradiation (day 2), mice are injected with
2.5.times.10.sup.4 RAJI cells via tail-vein. On day 3, the mice
(n=5 for each group) are injected by tail vein with 1 million
Pgp.sup.+, Pgp.sup.-, or unpurified T cells that had been infected
with the FMC63(vL-vH)-Myc-BBz-PAC lentivirus. Control mice (n=5)
are injected with RAJI cells but do not receive T cells. Survival
of mice injected with Pgp.sup.+ CAR-T cells is significantly higher
than those of mice injected with Pgp.sup.- CAR-T cells or
unpurified T cells. This is true irrespective of the method (flow
sorting, MACS or TH9402 plus light exposure, selection with
vincristine or hyperthermia) used to purify Pgp.sup.+ cells. PCR
analysis for the presence of CAR-modified T cells in blood and bone
marrow reveals longer in vivo persistence of CAR-T cells that are
derived from Pgp.sup.+ cells.
The above experiment is repeated using Pgp.sup.+/CD8.sup.+,
Pgp.sup.-/CD8.sup.+, Pgp.sup.+/CD4.sup.+, Pgp.sup.+/CD4.sup.-
starting population of cells. Again, CAR generated from Pgp+ve
cells perform better than those generated from Pgp.sup.- cells and
persisted longer in vivo.
Example 12. Use of Autologous Pgp-Expressing Cells for Adoptive
Cell Therapy
Patients with relapsed Acute lymphocytic Leukemia (ALL) or
high-risk intermediate grade B-cell lymphomas may receive
immunotherapy with adoptively transferred autologous Pgp.sup.+ T
cells-derived CAR-T cells. A leukapheresis product collected from
each patient undergoes selection of Pgp.sup.+ T cells using Flow
sorting with Pgp antibodies, MACS using Pgp antibodies,
Photodynamic selection following exposure to TH9402 plus light,
selection with vincristine or hyperthermia. Cells are transduced
with clinical grade CD19CAR virus and then selection and expansion
of the CAR-T cells occur in a closed system. After the resulting
cell products have undergone quality control testing (including
sterility and tumor specific cytotoxicity tests), they are
cryopreserved. Meanwhile, following leukapheresis, study
participants commence with lymphodepletive chemotherapy following
which they receive their cryopreserved CAR-T cells. The CAR-T cell
product is transported, thawed and infused at the patient's
bedside. The dose of CAR-T product varies from 1.times.10.sup.4
CAR.sup.+ CD3 cells/kg to 1.times.10.sup.9 CAR.sup.+ CD3 cells/kg
as per the study protocol. The CAR product may be administered in a
single infusion or split infusions. Research participants can be
pre-medicated at least 30 minutes prior to T cell infusion with 15
mg/kg of acetaminophen P.O. (max. 650 mg) and diphenhydramine 0.5-1
mg/kg I.V. (max dose 50 mg). Clinical and laboratory correlative
follow-up studies can then be performed at the physician's
discretion, and may include quantitative RT-PCR studies for the
presence of CD19-expressing ALL/lymphoma cells and/or the
adoptively transferred T cells; FDG-PET and/or CT scans; bone
marrow examination for disease specific pathologic evaluation;
lymph node biopsy; and/or long-term follow up per the guidelines
set forth by the FDA's Biologic Response Modifiers Advisory
Committee that apply to gene transfer studies.
Example 13. Use of Allogeneic Pgp-Expressing Cells for Adoptive
Cells Therapy
Patients with relapsed Acute Lymphocytic Leukemia (ALL) or
high-risk intermediate grade B-cell lymphomas who have undergone an
allogeneic bone marrow transplant may receive immunotherapy with
adoptively transferred allogeneic Pgp.sup.+ T cells-derived CAR-T
cells. A leukapheresis product collected from the donor (same donor
as used for the allogeneic transplant) undergoes selection of
Pgp.sup.+ T cells using Flow sorting following staining with Pgp
antibodies, MACS following staining with Pgp antibodies,
Photodynamic selection following exposure to TH9402 plus light,
selection with vincristine or hyperthermia. Cells are transduced
with clinical grade CD19-CAR and then selection and expansion of
the CAR-T cells occur in a closed system. After the resulting cell
products have undergone quality control testing (including
sterility and tumor specific cytotoxicity tests), they are
cryopreserved. Meanwhile, study participants commence with
lymphodepletive chemotherapy following which they receive the
cryopreserved allogeneic CAR-T cells. The CAR-T cell product is
transported, thawed and infused at the patient's bedside. The dose
of CAR-T product may vary from 1.times.10.sup.4 CAR.sup.+ CD3
cells/kg to 1.times.10.sup.9 CAR.sup.+ CD3 cells/kg as per the
study protocol. The CAR product may be administered in a single
infusion or split infusions. Research participants can be
pre-medicated at least 30 minutes prior to T cell infusion with 15
mg/kg of acetaminophen P.O. (max. 650 mg) and diphenhydramine 0.5-1
mg/kg I.V. (max dose 50 mg). Clinical and laboratory correlative
follow-up studies can then be performed at the physician's
discretion, and may include quantitative RT-PCR studies for the
presence of CD19-expressing ALL/lymphoma cells and/or the
adoptively transferred T cells; FDG-PET and/or CT scans; bone
marrow examination for disease specific pathologic evaluation;
lymph node biopsy; and/or long-term follow up per the guidelines
set forth by the FDA's Biologic Response Modifiers Advisory
Committee that apply to gene transfer studies. Use of
immunosuppressive drugs is also at the discretion of the
physician.
Example 14. Use of Pgp Negative T Cells to Reduce the Incidence of
GVHD in Patients Undergoing Allogeneic Bone Marrow Transplant and
Other Disorders
Peripheral blood stem cells are obtained from a donor using
leukopheresis following standard procedures. The donor may be, for
example, an HLA-matched (10/10 match) sibling donor, 10/10 matched
unrelated donor, or Antigen mismatched sibling or unrelated donor,
or a haploidentical donor. The leukopheresed product is enriched
for CD34-expressing cells by positive selection using the CliniMACS
Prodigy.RTM. System from Miltenyi Biotec and following the
manufacturer's recommendations. The CD34.sup.- fraction is labeled
with one or more antibodies against extracellular domain of Pgp
(e.g. UIC2, 4E3, MRK16 etc.) followed by incubation with Goat
anti-mouse IgG magnetic beads and negative selection using the
CliniMACS Prodigy.RTM. System, and following the manufacturer's
recommendations. Alternatively, negative selection for
Pgp-expressing cells can be obtained by using one, or a cocktail,
of Pgp antibodies that are directly conjugated to magnetic beads.
The Pgp negative fraction should contain less than 50%
Pgp-expressing cells, or preferably less than 40% Pgp-expressing
cells, or preferably less than 30% Pgp-expressing cells, or
preferably less than 20% Pgp-expressing cells, or preferably less
than 10% Pgp-expressing cells, or preferably less than 5%
Pgp-expressing cells, or preferably less than 1% Pgp-expressing
cells. The patient who has received the conditioning regimen
(myeloablative or reduced intensity or non-myeloablative) to
prepare for transplant is then administered by intravenous infusion
the CD34 enriched stem cell fraction along with Pgp-depleted T cell
fraction. The proportion and amount of Pgp-depleted T cell fraction
that is administered to the patient is at the discretion of the
physician. For example, from 1.times.10.sup.4 CD3 cells/kg to
1.times.10.sup.9 CD3 cells/kg may be infused either as a single
infusion or split infusion depending on the tolerance of the
patient and discretion of the treating physician.
Example 15. Use of Pgp-Negative T Cell Product for the Treatment of
CMV (Cytomegalovirus) Infection in an Allogeneic Stem Cell
Transplant Recipient
A patient who is status-post allogeneic stem cell transplant from
an unrelated donor develops refractory CMV infection. Peripheral
blood mononuclear cells are collected from the original donor, who
is CMV seropositive. T cells are first enriched by negative
selection using a cocktail of antibodies against non-T cell markers
and using the CliniMACS Prodigy.RTM. System. The T cell fraction is
then depleted of Pgp-expressing cells by incubation with a Pgp
antibody or cocktail of antibodies (1-2 .mu.g/million cells)
followed by negative selection using the CliniMACS Prodigy.RTM.
System, and following the manufacturer's recommendations. The
Pgp-depleted fraction of T cells is administered to the patient
intravenously either as a single infusion or in increasing
fractions, at the discretion of the treating physician. For
example, from 1.times.10.sup.4 Pgp.sup.-/CD3.sup.+ cells/kg to
1.times.10.sup.9 Pgp.sup.-/CD3.sup.+ cells/kg may be infused either
as a single infusion or split infusion depending on the tolerance
of the patient and discretion of the treating physician.
Example 16. Use of Pgp-Negative T Cell Product for the Treatment of
CMV (Cytomegalovirus) Infection in an Immunodeficient HIV/AIDS
Recipient
A patient with HIV/AIDS develops refractory CMV infection.
Peripheral blood mononuclear cells are collected from an HLA
matched donor, who is CMV seropositive. T cells are first enriched
by negative selection using a cocktail of antibodies against non-T
cell markers and using the CliniMACS Prodigy.RTM. System. The T
cell enriched fraction is then depleted of Pgp-expressing cells by
incubation with a Pgp antibody or a cocktail of antibodies (1-2
.mu.g/million cells) followed by negative selection using the
CliniMACS Prodigy.RTM. System, and following the manufacturer's
recommendations. The Pgp-depleted fraction of T cells is
administered to the patient intravenously either as a single
infusion or in increasing fractions, at the discretion of the
treating physician. For example, from 1.times.10.sup.4
Pgp.sup.-/CD3.sup.+ cells/kg to 1.times.10.sup.9
Pgp.sup.-/CD3.sup.+ cells/kg may be infused either as a single
infusion or split infusion depending on the tolerance of the
patient and discretion of the treating physician.
Example 17. Use of Pgp-Negative T Cell Product for the Treatment of
Adenovirus Infection in an Allogeneic Stem Cell Transplant
Recipient
A patient who is status-post allogeneic stem cell transplant from
an unrelated donor develops refractory adenovirus infection.
Peripheral blood mononuclear cells are collected from the original
donor, who is adenovirus seropositive. T cells are first enriched
by negative selection using a cocktail of antibodies against non-T
cell markers and using the CliniMACS Prodigy.RTM. System. The T
cell fraction is then depleted of Pgp-expressing cells by
incubation with a Pgp antibody or cocktail of antibodies (1-2
.mu.g/million cells) followed by negative selection using the
CliniMACS Prodigy.RTM. System, and following the manufacturer's
recommendations. The Pgp-depleted fraction of T cells is
administered to the patient intravenously either as a single
infusion or in increasing fractions, at the discretion of the
treating physician. For example, from 1.times.10.sup.4
Pgp.sup.-/CD3.sup.+ cells/kg to 1.times.10.sup.9
Pgp.sup.-/CD3.sup.+ cells/kg may be infused either as a single
infusion or split infusions depending on the tolerance of the
patient and discretion of the treating physician.
Example 18. Use of Pgp-Negative T Cell Product for the Treatment of
BK Virus Infection in an Allogeneic Stem Cell Transplant
Recipient
A patient who is status-post allogeneic stem cell transplant from
an unrelated donor develops refractory BK virus infection.
Peripheral blood mononuclear cells are collected from the original
donor BK virus seropositive. T cells are first enriched by negative
selection using a cocktail of antibodies against non-T cell markers
and using the CliniMACS Prodigy.RTM. System. The T cell fraction is
then depleted of Pgp-expressing cells by incubation with a Pgp
antibody or cocktail of antibodies (1-2 .mu.g/million cells)
followed by negative selection using the CliniMACS Prodigy.RTM.
System, and following the manufacturer's recommendations. The
Pgp-depleted fraction of T cells is administered to the patient
intravenously either as a single infusion or in increasing
fractions, at the discretion of the treating physician. For
example, from 1.times.10.sup.4 Pgp.sup.-/CD3.sup.+ cells/kg to
1.times.10.sup.9 Pgp.sup.-/CD3.sup.+ cells/kg may be infused either
as a single infusion or split infusion depending on the tolerance
of the patient and discretion of the treating physician.
Example 19. Enrichment of Pgp/CD34.sup.+ Pluripotent Hematopoietic
Stem Cells Using Serum-Starvation
The most primitive pluripotent hematopoietic stem cells are known
to co-express CD34 and Pgp and efflux DiOC2(3) (Chaudhary and
Roninson, Cell, 66, 85-94, 1991). To examine if these cells can be
enriched by serum starvation, peripheral blood stem cell cells were
collected by apheresis from a patient undergoing stem cell
transplantation after mobilization with chemotherapy and G-CSF
using standard procedures for stem cell mobilization and
collection. Cells underwent RBC lysis to get rid of red cells and
Ficoll-Hypaque separation to enrich for mononuclear cells. Cells
were cultured for 6 days in RPMI medium containing 2%, 5% or 10%
Fetal bovine Serum (FBS). Cells were stained with DiOC2(3) (60
ng/ml in 5 ml of RPMI 10% FBS medium at 4.degree. C. for 40 min).
The cells were washed with medium, dye-efflux in 10 ml RPMi 10%
medium at 37.degree. C. for 90 min, washed twice with PBS 1% FBS,
and stained with 1.5 .mu.l/sample/100 .mu.l of CD34-APCefluor 780
(ebiosciences cat#470349-42) at 4.degree. C. for 1 h. The cells
were washed and analyzed by Flow Cytometry. APC-efluor-780 was
detected in APC-Cy7 channel in BD Facsverse. FIG. 3 shows
significant enrichment of CD34.sup.+ stem cells from 4.6% to 5% to
7% upon reduction of FBS from 10% to 5% to 2%. Furthermore, there
was significant enrichment of the most primitive hematopoietic stem
cells that are DiOC2(3)-dull (or Pgp.sup.+) and CD34.sup.+ from
0.6% to 2% to 4% upon reduction of FBS from 10% to 5% to 2%. These
results demonstrate that CD34.sup.+ hematopoietic stem cells and
CD34.sup.+/Pgp.sup.+ pluripotent hematopoietic stem cells can be
enriched by serum starvation.
Example 20. Enrichment of Pgp.sup.+/CD34.sup.+ Pluripotent
Hematopoietic Stem Cells by Growth Factor-Starvation
The experiment was conducted as in the preceding example except
that cells were cultured for 6 days in XVIVO-10 (Lonza) medium
alone or XVIVO-10 medium containing SCF (50 ng/ml), TPO (50 ng/ml),
FLT3 (50 ng/ml), IL3 (50 ng/ml) and IL6 (50 ng/ml). There was an
enrichment of CD34.sup.+/DiOC2(3)-dull pluripotent hematopoietic
stem cells from 2% to 4% when the cells were cultured in the medium
lacking growth factors (FIG. 4).
Example 21. Use of Pgp-Positive/CD34.sup.+ Cell Product for
Allogeneic Stem Cell Transplantation
A patient with aplastic anemia is a candidate for allogeneic bone
marrow (or peripheral blood stem cell) transplant from a one
antigen mismatched related donor. Bone marrow is harvested from the
donor under general anesthesia. In order to reduce the incidence of
graft-vs-host disease, the bone marrow is enriched for
hematopoietic stem cells by positive selection for CD34.sup.+ cells
using CliniMACS Prodigy.RTM. System. To further enrich for the most
primitive hematopoietic progenitors, the CD34.sup.+ cell fraction
is enriched for cells that are Pgp.sup.+ by exposure to TH9402 plus
light, selection with vincristine or hyperthermia. For enrichment
using TH9402, cells are incubated at 37.degree. C. with 10 .mu.M
TH9402 (Theratechnologies, Montreal, QC, Canada) in X-Vivo 15
medium with 2.5% HAB. After a 40-minute incubation, cells are
centrifuged and dye efflux favored by resuspending cells in
TH9402-free medium for 90 minutes. At the end of the latter dye
efflux period, cells are exposed to a fluorescent light-scanning
device (PDCT-Xerox Series 4, Theratechnologies) delivering 5
J/cm.sup.2 at a wavelength of 514 nm. For enrichment using
hyperthermia, the cells are exposed to 43.degree. C. for 3 hours.
The final CD34.sup.+/Pgp.sup.+ cellular product (2.5-5 million
cells/Kg of body weight) is used for allogeneic transplantation
after the patient has received myeloablative conditioning regimen.
Patient receives standard post-allogeneic transplant care,
including use of immunosuppressive drugs, under the direction of
the treating physician.
Example 22. Use of Pgp-Positive/CD34+ Cell Product for Gone
Therapy
A patient with X-linked severe combined immunodeficiency (SCID-X1)
is a candidate for gene therapy with interleukin-2 receptor
.gamma.-chain (.gamma.c) complementary DNA. Bone marrow is
harvested from the patient under general anesthesia. Bone marrow is
enriched for hematopoietic stem cells by positive selection for
CD34.sup.+ cells using CliniMACS Prodigy.RTM. System. To further
enrich for the most primitive hematopoietic progenitors, the
CD34.sup.+ cell fraction is enriched for cells that are Pgp.sup.+
by exposure to TH9402 plus light, selection with vincristine or
hyperthermia. For enrichment using TH9402, cells are incubated at
37.degree. C. with 10 .mu.M TH9402 (Theratechnologies, Montreal,
QC, Canada) in X-Vivo 15 medium with 2.5% HAB. After a 40-minute
incubation, cells are centrifuged and dye efflux favored by
resuspending cells in TH9402-free medium for 90 minutes. At the end
of the latter dye efflux period, cells are exposed to a fluorescent
light-scanning device (PDCT-Xerox Series 4, Theratechnologies)
delivering 5 J/cm.sup.2 at a wavelength of 514 nm. For enrichment
using hyperthermia, the cells are exposed to 42.5.degree. C. for 3
hours. The final CD34.sup.+/Pgp.sup.+ cellular product (5 million
cells/Kg of body weight) is used for gene transfer with a
lentiviral vector encoding the .gamma.c cDNA using published
regimen (Gaspar H B et al., Lancet, 2004; 364:2181-7). Patient
receives infusion of approximately 5.times.10.sup.6/Kg
CD34.sup.+/Pgp.sup.+ gene modified stem cells without preparative
conditioning. Patient showed sustained recovery of T cells,
including CD3.sup.+, CD8.sup.+ and CD4.sup.+ subsets and normal
immunological function.
Example 23. Enrichment of Stem Like T Cells Using
Serum-Starvation
To examine if T stem cells can be enriched by serum starvation, the
experiment in example 19 is repeated using T cells isolated from
peripheral blood. There is enrichment of the DiOC2(3)-dull (or
Pgp+) cells following culture in RPMI medium containing 2% FCS as
compared to RPMI medium containing 10% serum. These results
demonstrate that T cells with stem like characteristic and/or
Pgp.sup.+ T cells can be enriched by serum starvation.
A number of embodiments have been set forth above to illustrate the
invention. The following claims further set forth what the
Applicants regard as their invention.
SEQUENCE LISTINGS
1
119659DNAArtificial SequenceVector -
pLenti-EF1a-FMC63(vL-vH)-Myc-BBz-T2A- Pac-A13 1aatgtagtct
tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa
ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga
tacataaacg ggtctctctg 240gttagaccag atctgagcct gggagctctc
tggctaacta gggaacccac tgcttaagcc 300tcaataaagc ttgccttgag
tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360taactagaga
tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
420aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
gactcggctt 480gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
tgagtacgcc aaaaattttg 540actagcggag gctagaagga gagagatggg
tgcgagagcg tcagtattaa gcgggggaga 600attagatcgc gatgggaaaa
aattcggtta aggccagggg gaaagaaaaa atataaatta 660aaacatatag
tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
720gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
tcagacagga 780tcagaagaac ttagatcatt atataataca gtagcaaccc
tctattgtgt gcatcaaagg 840atagagataa aagacaccaa ggaagcttta
gacaagatag aggaagagca aaacaaaagt 900aagaccaccg cacagcaagc
ggccgctgat cttcagacct ggaggaggag atatgaggga 960caattggaga
agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
1020acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
gaataggagc 1080tttgttcctt gggttcttgg gagcagcagg aagcactatg
ggcgcagcgt caatgacgct 1140gacggtacag gccagacaat tattgtctgg
tatagtgcag cagcagaaca atttgctgag 1200ggctattgag gcgcaacagc
atctgttgca actcacagtc tggggcatca agcagctcca 1260ggcaagaatc
ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
1320ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
ggagtaataa 1380atctctggaa cagatttgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1440ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1500acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1560ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
1620agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
cattatcgtt 1680tcagacccac ctcccaaccc cgaggggacc cgacaggccc
gaaggaatag aagaagaagg 1740tggagagaga gacagagaca gatccattcg
attagtgaac ggatctcgac ggtatcggtt 1800aacttttaaa agaaaagggg
ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860aatagcaaca
gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt
1920atcgataagc tttgcaaaga tggataaagt tttaaacaga gaggaatctt
tgcagctaat 1980ggaccttcta ggtcttgaaa ggagtgcctc gtgaggctcc
ggtgcccgtc agtgggcaga 2040gcgcacatcg cccacagtcc ccgagaagtt
ggggggaggg gtcggcaatt gaaccggtgc 2100ctagagaagg tggcgcgggg
taaactggga aagtgatgtc gtgtactggc tccgcctttt 2160tcccgagggt
gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
2220caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
ggcctggcct 2280ctttacgggt tatggccctt gcgtgccttg aattacttcc
acctggctgc agtacgtgat 2340tcttgatccc gagcttcggg ttggaagtgg
gtgggagagt tcgaggcctt gcgcttaagg 2400agccccttcg cctcgtgctt
gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg 2460aatctggtgg
caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa
2520tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
atgcgggcca 2580agatctgcac actggtattt cggtttttgg ggccgcgggc
ggcgacgggg cccgtgcgtc 2640ccagcgcaca tgttcggcga ggcggggcct
gcgagcgcgg ccaccgagaa tcggacgggg 2700gtagtctcaa gctggccggc
ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc 2760gccctgggcg
gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct
2820tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
agcgggcggg 2880tgagtcaccc acacaaagga aaagggcctt tccgtcctca
gccgtcgctt catgtgactc 2940cacggagtac cgggcgccgt ccaggcacct
cgattagttc tcgacctttt ggagtacgtc 3000gtctttaggt tggggggagg
ggttttatgc gatggagttt ccccacactg agtgggtgga 3060gactgaagtt
aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga
3120gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
ttcttccatt 3180tcaggtgtcg tgaggaatta gcttggtact aatacgactc
actataggga gacccaagct 3240ggctagttaa gcttgatatc gaattcctgc
agcccggggg atctgctagc atggccttac 3300cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg ccggacatcc 3360agatgacaca
gactacatcc tccctgtctg cctctctggg agacagagtc accatcagtt
3420gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa
ccagatggaa 3480ctgttaaact cctgatctac catacatcaa gattacactc
aggagtccca tcaaggttca 3540gtggcagtgg gtctggaaca gattattctc
tcaccattag caacctggag caagaagata 3600ttgccactta cttttgccaa
cagggtaata cgcttccgta cacgttcgga ggggggacca 3660agctggagat
cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg
3720aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc
ctgtccgtca 3780catgcactgt ctcaggggtc tcattacccg actatggtgt
aagctggatt cgccagcctc 3840cacgaaaggg tctggagtgg ctgggagtaa
tatggggtag tgaaaccaca tactataatt 3900cagctctcaa atccagactg
accatcatca aggacaactc caagagccaa gttttcttaa 3960aaatgaacag
tctgcaaact gatgacacag ccatttacta ctgtgccaaa cattattact
4020acggtggtag ctatgctatg gactactggg gtcaaggaac ctcagtcacc
gtctcctcac 4080gcgtagagca gaaactgatc tcggaagagg atctggcgaa
gcccaccacg acgccagcgc 4140cgcgaccacc aacaccggcg cccaccatcg
cgtcgcagcc cctgtccctg cgcccagagg 4200cgtgccggcc agcggcgggg
ggcgcagtgc acacgagggg gctggacttc gcctgtgaca 4260tctacatctg
ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca
4320ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa
ccatttatga 4380gaccagtaca aactactcaa gaggaagatg gctgtagctg
ccgatttcca gaagaagaag 4440aaggaggatg tgaactgaga gtgaagttca
gcaggagcgc agacgccccc gcgtaccagc 4500agggccagaa ccagctctat
aacgagctga atctaggacg aagagaggag tacgatgttt 4560tggacaagag
acgtggccgg gaccctgaga tggggggaaa gccgagaagg aagaaccctc
4620aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac
agtgagattg 4680ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg
cctttaccag ggtctcagta 4740cagccaccaa ggacacctac gacgcccttc
acatgcaggc cctgccccct cgctctagtg 4800gctccggcga gggcagagga
agtctactaa cctgcggaga tgtggaagaa aatcctggcc 4860cacatatgac
cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
4920ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
accgtcgatc 4980cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
cttcctcacg cgcgtcgggc 5040tcgacatcgg caaggtgtgg gtcgcggacg
acggcgccgc ggtggcggtc tggaccacgc 5100cggagagcgt cgaagcgggg
gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga 5160gcggttcccg
gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
5220aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
ggcaagggtc 5280tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
gcgcgccggg gtgcccgcct 5340tcctggagac ctccgcgccc cgcaacctcc
ccttctacga gcggctcggc ttcaccgtca 5400ccgccgacgt cgaggtgccc
gaaggaccgc gcacctggtg catgacccgc aagcccggtg 5460cctgagtcga
ctccggatga tcagggccct gtacagatat cgacaatcaa cctctggatt
5520acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
acgctatgtg 5580gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
ccgtatggct ttcattttct 5640cctccttgta taaatcctgg ttgctgtctc
tttatgagga gttgtggccc gttgtcaggc 5700aacgtggcgt ggtgtgcact
gtgtttgctg acgcaacccc cactggttgg ggcattgcca 5760ccacctgtca
gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
5820tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
actgacaatt 5880ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
gctcgcctgt gttgccacct 5940ggattctgcg cgggacgtcc ttctgctacg
tcccttcggc cctcaatcca gcggaccttc 6000cttcccgcgg cctgctgccg
gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 6060cgagtcggat
ctccctttgg gccgcctccc cgcctggaat tcgagctcgg tacctttaag
6120accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
aggggggact 6180ggaagggcta attcactccc aacgaagaca agatctgctt
tttgcttgta ctgggtctct 6240ctggttagac cagatctgag cctgggagct
ctctggctaa ctagggaacc cactgcttaa 6300gcctcaataa agcttgcctt
gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 6360tggtaactag
agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
6420ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
cagagagtga 6480gaggaacttg tttattgcag cttataatgg ttacaaataa
agcaatagca tcacaaattt 6540cacaaataaa gcattttttt cactgcattc
tagttgtggt ttgtccaaac tcatcaatgt 6600atcttatcat gtctggctct
agctatcccg cccctaactc cgcccatccc gcccctaact 6660ccgcccagtt
ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
6720gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
ttttggaggc 6780ctagggacgt acccaattcg ccctatagtg agtcgtatta
cgcgcgctca ctggccgtcg 6840ttttacaacg tcgtgactgg gaaaaccctg
gcgttaccca acttaatcgc cttgcagcac 6900atcccccttt cgccagctgg
cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6960agttgcgcag
cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
7020gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
cccgctcctt 7080tcgctttctt cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc 7140gggggctccc tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg 7200attagggtga tggttcacgt
agtgggccat cgccctgata gacggttttt cgccctttga 7260cgttggagtc
cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
7320ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
tattggttaa 7380aaaatgagct gatttaacaa aaatttaacg cgaattttaa
caaaatatta acgcttacaa 7440tttaggtggc acttttcggg gaaatgtgcg
cggaacccct atttgtttat ttttctaaat 7500acattcaaat atgtatccgc
tcatgagaca ataaccctga taaatgcttc aataatattg 7560aaaaaggaag
agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
7620attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
atgctgaaga 7680tcagttgggt gcacgagtgg gttacatcga actggatctc
aacagcggta agatccttga 7740gagttttcgc cccgaagaac gttttccaat
gatgagcact tttaaagttc tgctatgtgg 7800cgcggtatta tcccgtattg
acgccgggca agagcaactc ggtcgccgca tacactattc 7860tcagaatgac
ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
7920agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
ccaacttact 7980tctgacaacg atcggaggac cgaaggagct aaccgctttt
ttgcacaaca tgggggatca 8040tgtaactcgc cttgatcgtt gggaaccgga
gctgaatgaa gccataccaa acgacgagcg 8100tgacaccacg atgcctgtag
caatggcaac aacgttgcgc aaactattaa ctggcgaact 8160acttactcta
gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
8220accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
ctggagccgg 8280tgagcgtggg tctcgcggta tcattgcagc actggggcca
gatggtaagc cctcccgtat 8340cgtagttatc tacacgacgg ggagtcaggc
aactatggat gaacgaaata gacagatcgc 8400tgagataggt gcctcactga
ttaagcattg gtaactgtca gaccaagttt actcatatat 8460actttagatt
gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
8520tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
cgtcagaccc 8580cgtagaaaag atcaaaggat cttcttgaga tccttttttt
ctgcgcgtaa tctgctgctt 8640gcaaacaaaa aaaccaccgc taccagcggt
ggtttgtttg ccggatcaag agctaccaac 8700tctttttccg aaggtaactg
gcttcagcag agcgcagata ccaaatactg ttcttctagt 8760gtagccgtag
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
8820gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
ccgggttgga 8880ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
tgaacggggg gttcgtgcac 8940acagcccagc ttggagcgaa cgacctacac
cgaactgaga tacctacagc gtgagctatg 9000agaaagcgcc acgcttcccg
aagggagaaa ggcggacagg tatccggtaa gcggcagggt 9060cggaacagga
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
9120tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
caggggggcg 9180gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
ttcctggcct tttgctggcc 9240ttttgctcac atgttctttc ctgcgttatc
ccctgattct gtggataacc gtattaccgc 9300ctttgagtga gctgataccg
ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 9360cgaggaagcg
gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
9420ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
gcaacgcaat 9480taatgtgagt tagctcactc attaggcacc ccaggcttta
cactttatgc ttccggctcg 9540tatgttgtgt ggaattgtga gcggataaca
atttcacaca ggaaacagct atgaccatga 9600ttacgccaag cgcgcaatta
accctcacta aagggaacaa aagctggagc tgcaagctt 9659
* * * * *